Targeting TROP2xPD-L1 bispecific antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention relates to an antibody specifically binding to TROP2 or an antigen binding fragment thereof. The invention also encompasses VHH antibodies or heavy chain antibodies that specifically bind to PD-L1. The invention further relates to a bispecific antibody which is constructed on the basis of the TROP2 antibody and the PD-L1 antibody and is specifically combined with the TROP2 and the PD-L1. The invention also encompasses immunoconjugates constructed based on the antibody molecules, such as antibody-conjugated drugs such as ADC. The invention further relates to a preparation method and application of the antibody molecule and the TROP2xPD-L1 targeting bispecific antibody coupling medicine.

Description

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410524183.0, filed on April 28, 2024, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to an antibody that specifically binds to TROP2 or an antigen-binding fragment thereof. The invention also covers VHH antibodies or heavy chain antibodies that specifically bind to PD-L1. The invention further relates to bispecific antibodies specifically binding to TROP2 and PD-L1 constructed based on TROP2 or PD-L1 antibodies. The invention also covers immunoconjugates constructed based on said antibody molecules, such as antibody-drug conjugates (ADCs). The invention also relates to methods for preparing antibody molecules and bispecific antibody-drug conjugates targeting TROP2xPD-L1, and their applications. Background of the Invention

[0004] Antibody-drug conjugates (ADCs) typically consist of three parts: an antibody, a payload (e.g., a bioactive small molecule), and a linker. The payload is covalently coupled to the antibody via a linker; the antibody (e.g., a monoclonal antibody) specifically recognizes a specific target on the surface of tumor cells, thereby guiding the ADC to the surface of cancer cells and allowing the ADC to enter the cancer cells via endocytosis; then, the payload is released in the tumor microenvironment, thereby specifically killing cancer cells without damaging normal tissue.

[0005] TROP2, a trophoblast cell surface antigen also known as tumor-associated calcium signaling transducer 2 (TACSTD2), is overexpressed in various human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer (10.3389 / fonc.2022.951589). PD-L1, also known as programmed death-ligand 1, plays a crucial role in cancer therapy. First, PD-L1 is expressed on the surface of various cells, including many tumor cells and immune cells. The interaction between PD-L1 and its receptor PD-1 on immune cells has been found to be essential for regulating the immune response. When PD-L1 binds to PD-1, it sends an inhibitory signal to suppress the activity of T cells, which are crucial for fighting cancer cells. This interaction helps tumors evade immune surveillance and promotes their growth. Immune checkpoint inhibitor drugs aim to block the interaction between PD-L1 and PD-1, thereby unblocking the immune system and reactivating the T cell response against the tumor. By blocking this pathway, immune checkpoint inhibitors enhance anti-tumor immunity and can lead to durable responses in various types of cancer. They have shown significant success in treating malignancies such as melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and head and neck squamous cell carcinoma. By targeting the interaction between PD-L1 and its receptor PD-1, they restore T-cell-mediated anti-tumor immunity, thus revolutionizing cancer treatment. They provide new treatment options for patients with various malignancies and significantly improve prognosis in some cancers.

[0006] Assessing PD-L1 expression levels in tumor tissues has become an important biomarker for predicting response to immunotherapy. High levels of PD-L1 expression are often associated with better response rates to immune checkpoint inhibitors.

[0007] PD-L1 is a tumor-associated antigen, and its overexpression in various cancers is associated with immune escape mechanisms and tumor progression. Due to its highly specific expression on the surface of tumor cells, PD-L1 has been widely used as a target for antibody drugs in combination with targeted drugs and immunotherapy strategies.

[0008] Furthermore, one potential application is targeting PD-L1 as an antibody-drug conjugate (ADC). Using PD-L1 as an ADC target allows for personalized treatment of patients with high PD-L1 expression levels in cancer cells. For example, in non-small cell lung cancer (NSCLC), using a PD-L1 monoclonal antibody ADC with a toxin load can selectively kill tumor cells that highly express PD-L1, thereby improving treatment efficacy and reducing damage to normal tissues. Currently, data indicate that PD-L1 ADCs have shown promising anti-tumor activity in preclinical and clinical settings. For instance, PF-08046054 (SGN-PDL1V) has achieved good therapeutic effects in the clinical treatment of HNSCC (NCT05208762). In addition, another PD-L1 ADC, HLX-43, has also shown strong anti-tumor effects in preclinical trials. In summary, targeting PD-L1 as an ADC target can improve treatment efficacy by selectively killing tumor cells that highly express this molecule and holds promise as a personalized treatment strategy.

[0009] Preclinical and clinical studies have demonstrated the efficacy of anti-TROP2 antibody-drug conjugates (ADCs), such as Trodelvy, an anti-human TROP2 antibody-SN-38 conjugate, in cancer treatment. Clinical results show promising therapeutic effects of Trodelvy in treating refractory solid tumors. Trodelvy achieved an objective response rate (ORR) of 33% in patients with drug-resistant triple-negative breast cancer (TNBC). ADC is one of the key mechanisms of Trodelvy; the payload SN-38 is linked via a pH-sensitive linker. In the acidic tumor microenvironment, the linker breaks down, specifically releasing SN-38. However, because the Trodelvy linker is not stable, the maleimide-mediated linker breaks down under physiological conditions via thiol exchange, resulting in a relatively short serum half-life (approximately 1 day). Therefore, Trodelvy may have a relatively high off-target effect.

[0010] Furthermore, the combination of ADC and I / O (PD1 / PD-L1 antibody) is currently challenging first-line treatment options in various tumors (NSCLC, TNBC, etc.), clearly demonstrating the synergistic effect of the combination of ADC drugs and I / O.

[0011] Therefore, there is a need in the field to develop new antibodies that can target TROP2 and / or PD-L1, especially bispecific antibodies that can simultaneously target TROP2 and PD-L1, and then develop corresponding antibody-drug conjugates based on them. Summary of the Invention

[0012] One aspect of the present invention relates to a binding molecule that specifically binds to TROP2, such as an anti-TROP2 antibody or an antigen-binding fragment thereof, or a bispecific antibody constructed based thereon.

[0013] Another aspect of the invention relates to a VHH or heavy chain antibody that specifically binds to PD-L1, or a bispecific antibody constructed based thereon.

[0014] Another aspect of the invention relates to a multispecific binding molecule that specifically binds to TROP2 and PD-L1, such as a multispecific antibody, or a bispecific antibody. In some embodiments, the bispecific antibody targets both TROP2 and PD-L1 to reach more cancer patients and produce better cancer treatment outcomes.

[0015] The present invention also relates to immune conjugates, such as antibody-drug conjugates (ADCs), constructed based on the anti-TROP2 antibody or its antigen-binding fragment thereof, anti-PD-L1 VHH or heavy chain antibody, or the bispecific antibody of the present invention that specifically binds TROP2 and PD-L1.

[0016] This invention provides an immunoconjugate comprising an antibody targeting TROP2 and / or PD-L1 (e.g., the antibody of this invention that specifically binds to TROP2 and / or PD-L1 or its antigen-binding fragment) and other active agents. In some embodiments, the immunoconjugate of this invention comprises said antibody molecule and a payload. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0017] Therefore, the present invention provides a bispecific antibody targeting TROP2 and PD-L1 and an antibody-drug conjugate (ADC) targeting TROP2 and PD-L1, wherein the antibody-drug conjugate of the present invention has one or more of the following properties:

[0018] i) It can effectively recognize TROP2-positive tumor cells through the TROP2 terminus and kill tumors through ADC;

[0019] ii) The PD-L1 antibody in the molecule can also recognize the PD-L1 target on the surface of the tumor, and enter the cell through endocytosis-mediated toxin entry, thereby inhibiting the growth of tumor cells;

[0020] iii) The PDL1 in the molecule still maintains good PD-L1 blocking activity, which activates T cells in the immune system by blocking the interaction between PD-L1 and PD1, thereby further killing tumors;

[0021] iv) The tumor-killing effect of ADC induces immune cell death (the release of tumor-specific antigens to activate the immune system), which can synergize with the PD-L1 blocking effect in the molecule, thereby achieving better tumor treatment results. Attached Figure Description

[0022] Figure 1 : Detection of the endocytic activity of chimeric antibody chAb1 on FaDu cells;

[0023] Figure 2 : Detection of the endocytic activity of humanized antibody hzAb1.4 on FaDu cells;

[0024] Figure 3 : PD-L1 antibody humanization post-endocytic activity test;

[0025] Figure 4 Schematic diagram of the TROP2xPD-L1 dual antibody ADC bsAb-NT3 structure;

[0026] Figure 5 Detection of PD-L1 blocking activity of TROP2xPD-L1 bispecific antibody bsAb;

[0027] Figure 6 The endocytic activity of TROP2xPD-L1 bispecific antibody bsAb was detected on EBC1, BxPC3, HCC1954 and PANC-0813, respectively.

[0028] Figure 7 Cytotoxicity tests of TROP2xPD-L1 bispecific antibody ADCs bsAb-NT3 and hRS7-NT3 on EBC1, BxPC3, HCC1954 and PANC-0813, respectively;

[0029] Figure 8 : TROP2xPD-L1 bispecific antibody ADC bsAb-NT3 bystander effect test; A is the test of TROP2 and PD-L1 expression on the surface of Colo205 and HCC1954 cells. B is the bystander effect test of each ADC on insensitive Colo205 cells.

[0030] Figure 9 : Efficacy testing of ADC in the in vivo pancreatic cancer tumor model BxPC3;

[0031] Figure 10 : Efficacy testing of ADC in the in vivo non-small cell lung cancer tumor model H322;

[0032] Figure 11: Efficacy testing of ADC in the in vivo breast cancer tumor model HCC1954; A is the low-dose group, B is the high-dose group;

[0033] Figure 12 : Efficacy testing of ADC in the in vivo breast cancer tumor model HCC1954-hPDL1 (high expression of PD-L1); A is a schematic diagram of expression, B is the efficacy test;

[0034] Figure 13 : Efficacy testing of ADC in the mouse syngeneic colon cancer tumor model CT26-hTROP2-hPDL1; A is a schematic diagram of efficacy testing, B is a schematic diagram of survival testing;

[0035] Figure 14 : PK detection of ADC in mice; A is a schematic diagram of PK detection, and B is the result of ADC stability detection in vivo. Invention Details

[0036] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0037] I. Definition

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0040] The term “about” when used in conjunction with a numeric value means a range of numeric values ​​that have a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) smaller than the specified numeric value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) larger than the specified numeric value.

[0041] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0042] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. 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 that specific sequence.

[0043] When “first” and “second” are mentioned in this article, it is only to distinguish between two structural domains or two chains, and does not indicate the location of the two structural domains in any way.

[0044] As used herein, the terms "anti-TROP2 antibody," "anti-TROP2," "TROP2 antibody," or "antibody against TROP2" refer to antibodies that, or antigen-binding fragments thereof, bind to the TROP2 protein with sufficient affinity. These antibodies can be used as diagnostic and / or therapeutic agents targeting TROP2, or for constructing immunoconjugates, such as antibody-drug conjugates. In some embodiments of the invention, TROP2 is human TROP2. In some embodiments, TROP2 is the protein listed under UniProt accession number P09758. In some aspects, the anti-TROP2 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to other TROP2 target antigens, such as bispecific antibodies.

[0045] As used herein, the terms "anti-PD-L1 antibody," "anti-PD-L1," "PD-L1 antibody," or "antibody against PD-L1" refer to antibodies that, or antigen-binding fragments thereof, are capable of binding to the PD-L1 protein with sufficient affinity. These antibodies can be used as diagnostic and / or therapeutic agents targeting PD-L1, or for constructing immunoconjugates, such as antibody-drug conjugates. In some embodiments of the invention, PD-L1 is human PD-L1. In some embodiments, PD-L1 is a protein under accession number (Gene ID: 29126). In some aspects, the anti-PD-L1 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to other target antigens of PD-L1, such as bispecific antibodies.

[0046] When this article refers to "anti-TROP2 antibody" or "anti-PD-L1 antibody", it does not exclude the inclusion of multispecific antibodies that specifically bind to TROP2 and PD-L1, such as bispecific antibodies.

[0047] As used herein, the terms "anti-TROP2xPD-L1 bispecific antibody," "bispecific antibody that specifically binds to TROP2 and PD-L1," "anti-TROP2xPD-L1 antibody," or "TROP2xPD-L1 bispecific antibody" refer to antibodies that can bind to both the TROP2 and PD-L1 proteins with sufficient affinity. These antibodies can be used as diagnostic and / or therapeutic agents targeting TROP2 and / or PD-L1, or for constructing immunoconjugates, such as antibody-drug conjugates.

[0048] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2, and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0049] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0050] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in a particular amino acid being replaced by a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, conserved modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity to the target antigen relative to the parent antibody or binding protein.

[0051] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0052] When it is mentioned that "Ab is derived from antibody", it means that the binding domain of the Ab is or is derived from the binding domain of the antibody's specific binding antigen. For example, the specific binding antigen fragment of the Ab, such as Fab, is or is derived from the corresponding fragment of the antibody, such as Fab. Or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody. Or one, two, three, four, five or six CDRs of the antigen binding region are the CDRs of the antibody.

[0053] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0054] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0055] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing an immunoglobulin heavy chain variable domain (VH), a heavy chain constant domain (CH1), a light chain variable domain (VL), and a light chain constant domain (CL). One polypeptide chain contains VH and a constant region selected from CH1 and CL from its N-terminus to its C-terminus, while the other polypeptide chain contains VL and another constant region selected from CL and CH1 from its N-terminus to its C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0056] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (available at imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0057] In some embodiments, the CDR of the heavy chain variable region of the antibody in this invention is defined according to the Kabat or Chothia scheme. In some embodiments, the CDR of the light chain variable region of the antibody in this invention is determined according to the Kabat scheme.

[0058] In one embodiment, HCDR1 in the anti-TROP2 or PD-L1 antibody of the present invention is defined according to the Kabat and Chothia schemes, HCDR2 and HCDR3 are defined according to the Kabat schemes respectively, and LCDRs are defined according to the Kabat schemes respectively:

[0059]

[0060] The following is a calculation of sequence identity between sequences.

[0061] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0062] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portion of the variable region with the corresponding portion of the human antibody).

[0063] As used herein, the terms “anti,” “binding,” or “specific binding” mean that the binding interaction is selective for the target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0064] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (K). D This indicates that the dissociation constant is the dissociation rate constant and the association rate constant (Kdissociation and Kassociation, respectively). dis and K on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0065] The terms “VHH”, “VHH antibody”, or “single-domain antibody” are used interchangeably herein and generally refer to an antibody that contains only one heavy chain variable region or is composed of such a region and has antigen-binding activity. A VHH typically contains three CDRs and four highly conserved framework regions, and generally has the following structure: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4; and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme described in the “Definitions” section, preferably by using the Kabat, Chothia, or Kabat and Chothia schemes to define the boundaries of the three CDRs in the variable region sequence. VHHs typically consist only of heavy chain variable domains derived from heavy chain antibodies lacking light chains, also known as nanobodies. The VHH used in this invention is preferably derived from camel species, such as alpacas, or from their humanized or sequence-optimized forms (e.g., affinity-matured forms to increase binding affinity). In some embodiments, the VHH of this invention is a monovalent, monospecific polypeptide molecule consisting of or substantially consisting of a single heavy chain variable region (e.g., the heavy chain variable region of a heavy chain antibody).

[0066] The single-domain antibody or VHH of the present invention may also be contained in a larger polypeptide / protein. Examples of polypeptides / proteins containing the VHH of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or multispecific antibodies or immunoconjugates such as ADC molecules.

[0067] The "heavy chain antibody" described in this invention refers to an antibody that does not have a light chain. For example, its N-to-C segment may contain VH-Fc, VH-CH2-CH3, or VH-hinge region-CH2-CH3, or it may contain VH-CH1-CH2-CH3. The heavy chain antibody of this invention can also encompass homodimers, such as heavy chain dimer antibodies that do not have a light chain. The heavy chain antibody may contain VH from a standard antibody or VH from a single-domain antibody. For example, the VH in a heavy chain antibody may be VHH. In some embodiments, the heavy chain antibody of this invention may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from camel (lamb, camel, especially alpaca), in its humanized form or its sequence-optimized form (affinity-matured form), or a fragment thereof (e.g., a fragment containing at least a portion of the constant region). The heavy chain antibodies of the present invention also cover antibodies formed by fusing a heavy chain variable region or VHH with an Fc region (e.g., the Fc region of human IgG, such as the Fc region of human IgG1, IgG2, IgG3 or IgG4).

[0068] When “VHH” is mentioned in the context of heavy chain antibodies, multispecific antibodies, or fusion proteins, it should be understood that it is part of a multispecific antibody, not as a separate molecule.

[0069] The term "target" refers to the substance to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the present invention relates to a target-binding region derived from an antibody, "target" and "antigen" may be used interchangeably.

[0070] As used herein, the term "target-binding region" refers to the portion of a binding molecule, such as a multispecific or bispecific binding molecule, that binds to a specific target or antigen. A target-binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target-binding region may or may not have a tertiary structure independent of the remaining portion of the binding molecule and can bind to its target as a standalone entity or not. A target-binding region can also be a receptor or ligand, or a ligand-binding domain of a receptor. In the case of multispecific or bispecific antibodies, the "target-binding region" is also referred to as the "antigen-binding region."

[0071] As used herein, the term "antigen-binding region" refers to any portion of an antibody or its antigen-binding fragment, such as a multispecific or bispecific antibody, that binds to a specific target or antigen. An antigen-binding region can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remaining portion of the multispecific or bispecific antibody and may bind or not bind its antigen / epitope as a standalone entity. When the binding molecule of the present invention relates to a target-binding region derived from an antibody, the terms "target-binding region" and "antigen-binding region" may be used interchangeably.

[0072] As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigens or epitopes.

[0073] The term "bispecific antibody" refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope and the second antigen-binding region binds to another antigen or another epitope. Therefore, the bispecific antibody according to the invention comprises specificity for two different antigens, or for two different epitopes of one antigen. Bispecific antibody forms include IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8:130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, the heavy and light chains of each Fab potentially derived from separate monoclonal antibodies. The bispecific antibody of the invention can be prepared using bispecific antibody forms or techniques known in the art. Specific exemplary bispecific forms that can be used in the context of the invention can be found, for example, Labrijn et al., Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8):1-24.

[0074] When "first antigen-binding region" is mentioned in multispecific or bispecific antibodies, it refers to the binding region that binds to the first antigen or the first epitope, and there is no intention to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific or bispecific antibody may contain one or more first antigen-binding regions. For instance, a bispecific antibody contains both a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0075] When it is mentioned that "the target or antigen-binding region is derived from the antibody", it means that the binding domains constituting the target / antigen-binding region are or derived from the binding domains of the antibody that specifically bind antigens. For example, the specific antigen-binding fragment of the antigen-binding region, such as Fab, is or is derived from the corresponding fragment of the antibody, such as Fab. Or the heavy chain variable region and / or light chain variable region of the antigen-binding region are or are derived from the heavy chain variable region and / or light chain variable region of the antibody. Or one, two, three, four, five, or six CDRs of the antigen-binding region are the CDRs of the antibody.

[0076] The term "derived from" means that the fragment in the antigen-binding region is substantially identical to the fragment from the antibody from which it originated, but has mutations at one or more sites, such as substitution, deletion, or addition. In one specific embodiment, the mutation is not in the antibody's CDR.

[0077] The multispecific antibodies or heavy chain antibodies of the present invention may contain linkers. As used herein, the term "linker" refers to any molecule that enables direct linkage between different portions of a multispecific antibody. Examples of linkers that establish covalent links between different portions of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to a sequence of amino acids that links the amino acid sequences of the various portions of the multispecific antibody together. Preferably, the peptide linker has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other without impeding the desired activity. The peptide linker may primarily comprise or may not primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. Useful connectors include glycine-serine polymers, including, for example, (G)n, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). Useful connectors also include glycine-alanine polymers, alanine-serine polymers, and other flexible connectors. Exemplary connectors include sequences such as those shown in SEQ ID NO:29 or 51.

[0078] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. In this document, the terms “Fc region,” “Fc portion,” and “dimeric Fc (e.g., heterodimeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the Fc region refers to the region extending from Asp221 to the carboxyl terminus of the heavy chain. In one embodiment, the Fc region is derived from a human Fc region. The antibody Fc region directly participates in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to human IgG1, IgG2, IgG3, or an IgG subclass. In one embodiment, the Fc region is derived from the Fc region of either human IgG1, IgG2, IgG3, or IgG4.

[0079] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises, or is composed of, the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 57). In some embodiments, CH1 may contain a partial hinge region. In some embodiments, the Fc region may contain a partial hinge region.

[0080] As used in this article, "antibody-drug conjugate (ADC)" refers to a structure obtained by linking an antibody to a (small molecule) drug.

[0081] The term "linker" refers to a structural segment that connects a drug (e.g., a small molecule drug) to an antibody. It should be understood that a linker has functional groups that can form bonds with functional groups of the antibody or its antigen-binding fragment before it is linked to the antibody or its antigen-binding fragment.

[0082] The term "linker-payload" refers to a payload, such as a drug (e.g., a small molecule drug), which is linked to a linker to form a compound.

[0083] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, for example, 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0084] The term "alkylene" refers to an alkyl group as defined above, but which is divalent, i.e. has two single bonds attached to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0085] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one double bond but no triple bonds. The alkenyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl groups.

[0086] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one triple bond. The alkynyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentyynyl, and hexynyl.

[0087] The terms “halogen” or “halogenated” refer to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0088] The term "haloalkyl" refers to an alkyl group as defined herein, which is substituted with one or more halogen groups. Haloalkyl groups may preferably be monohaloalkyl, dihaloalkyl, or polyhaloalkyl (including perhaloalkyl). Monohaloalkyl groups may contain one iodine, bromine, chlorine, or fluorine group in the alkyl group. Dihaloalkyl and polyhaloalkyl groups may contain two or more identical halogen atoms or combinations of different halogen groups in the alkyl group. Preferably, polyhaloalkyl groups contain at most 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0089] The term “haloalkenyl” refers to an alkenyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The term “haloynyl” refers to an ynyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The meaning of “halogenated” as defined for “haloalkyl” may apply to both “haloalkenyl” and “haloynyl”.

[0090] The term "polyol group" refers to an alkyl group as defined above containing a plurality of (e.g., 2-10, e.g., 3, 4, 5, 6, 7, or 8) hydroxyl groups, optionally containing one or more (e.g., 2, 3, or 4) other groups (e.g., amino, carbonyl). Non-limiting examples of "polyol group" include, for example... Wherein, the chiral center whose stereo configuration is not specified can be of R or S configuration, preferably.

[0091] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. The common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, the amino acid can be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln).

[0092] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0093] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugates of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0094] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0095] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0096] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug fraction (D) coupled to the Ab fraction described herein to the Ab fraction. In some embodiments described herein, the DAR may be determined by p in Formula I, for example, the DAR may be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug fraction (D) coupled to the Ab fraction described herein to the Ab fraction in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR or the measured DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules that contains ADC molecules having the same and / or different DAR values.

[0097] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0098] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0099] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0100] The term "drug" refers to organic compounds that can regulate biological processes, especially altering or preventing pathological processes.

[0101] The term "prodrug" refers to a chemically modified active or inactive compound that, after being administered to an individual, undergoes physiological processes in the body (such as hydrolysis and metabolism) to become an active drug. Techniques for manufacturing and using prodrugs are well known to those skilled in the art.

[0102] The term "small molecule drug" refers to low-molecular-weight organic compounds that can regulate biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 500 kDa. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0103] "Antitumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, especially small molecule cytotoxic agents or chemotherapeutic agents, such as camptothecin compounds such as those disclosed in WO2021 / 173773, WO2022180581, CN 102574866, ixotecan (a topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative); auristatin compounds such as monomethyl auristatin E (MMAE); or maytansine compounds such as the small molecule microtubule inhibitor DM1. It should be understood that antitumor compounds can be substituted with isotopes including but not limited to deuterium and tritium. For example, after substitution with deuterium, the carbon-hydrogen bond is replaced by a carbon-deuterium bond. Since the former is more stable than the latter, this substitution can directly affect the absorption, distribution, metabolism, and excretion properties of certain drugs, thereby improving the efficacy, safety, and tolerability of the drug. Therefore, the term "antitumor compound" in this application can cover compounds that are substituted with deuterium.

[0104] "Deuterated" means that hydrogen in a molecule is replaced by deuterium, for example, one or more hydrogens, such as 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) hydrogens are replaced by deuterium.

[0105] "Camptothecin-like compounds" refer to compounds that possess a camptothecin core structure (e.g., the pentaneous or tetraaneous structure of camptothecin) and exhibit antitumor activity. "Camptothecin-like compounds" is a commonly used term in medicinal chemistry. Based on the structure of a compound, those skilled in the art can readily determine whether a compound belongs to the camptothecin-like class.

[0106] "Aurestatin compounds" refer to compounds that have the aurestatin core structure and possess antitumor activity.

[0107] "Maytansine compounds" refer to compounds having a maytansine core structure and possessing antitumor activity. The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates (e.g., activates) an immune response. An immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0108] The term "effective amount" refers to such an amount or dose of the antibody molecule or immunoconjugate or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0109] "Therapeutic effective dose" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective dose is also a dose in which any toxic or harmful effects of the antibody molecule or immune conjugate or composition or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, the "therapeutic effective dose" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0110] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0111] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which foreign nucleic acids have been introduced, including the progeny of such cells.

[0112] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0113] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0114] "Isolated" antibodies or other molecules (e.g., ADC molecules) are antibodies or molecules that have been separated from components of their natural environment or the environment in which they are expressed. In some embodiments, the antibody or ADC molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0115] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0116] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages, or it can be metastatic.

[0117] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Tumor” encompasses solid tumors and hematologic malignancies as well as metastatic lesions. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used in this article.

[0118] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0119] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0120] The terms "drug combination" or "combination product" refer to non-fixed or fixed combinations, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibody molecules or immunoconjugates of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the antibodies or immunoconjugates of the present invention used in the drug combination, such as ADC molecules and other therapeutic agents, are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0121] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0122] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0123] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0124] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0125] II. Antibody molecules

[0126] II-1 The anti-TROP2 antibody or its antigen-binding fragment of the present invention

[0127] In some embodiments, the anti-TROP2 antibody of the present invention, or its antigen-binding fragment, specifically binds to TROP2 (e.g., human TROP2, rhesus monkey TROP2, or cynomolgus monkey TROP2). In some embodiments, the anti-TROP2 antibody of the present invention, or its antigen-binding fragment, has a binding affinity K for TROP2 (e.g., human TROP2, rhesus monkey TROP2, or cynomolgus monkey TROP2). D The value is less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5 nM, or between these values. In some embodiments, the binding affinity K of the anti-TROP2 antibody of the present invention or its antigen-binding fragment to TROP2 (e.g., human TROP2, rhesus monkey TROP2, or cynomolgus monkey TROP2) is... D The values ​​are greater than or equal to approximately 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, or 0.5 nM. In some embodiments, the antibody binding affinity of the present invention is determined by a thin-layer interferometry technique, such as ForteBio.

[0128] In some embodiments, the anti-TROP2 antibody of the present invention or its antigen-binding fragment can effectively bind to TROP2, such as human, cynomolgus monkey, or rhesus monkey TROP2.

[0129] In some embodiments, the anti-TROP2 antibody of the present invention or its antigen-binding fragment is internalized on TROP2-positive cells, such as TROP2-positive tumor cells, for example, with good internalization effect.

[0130] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0131] In some aspects, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VH). In some aspects, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises both a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0132] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region and a light chain constant region.

[0133] In some embodiments, the heavy chain variable region of the anti-TROP2 antibody or its antigen-binding fragment described in this invention:

[0134] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 1 or 23; or

[0135] (ii) Contains or is composed of an amino acid sequence selected from or consisting of the amino acid sequence shown in SEQ ID NO:1 or 23; or

[0136] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:1 or 23, preferably, the amino acid changes do not occur in the CDR region.

[0137] In some embodiments, the light chain variable region of the anti-TROP2 antibody or its antigen-binding fragment described in this invention...

[0138] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 6 or 24; or

[0139] (ii) Contains or is composed of an amino acid sequence selected from or consisting of the amino acid sequence shown in SEQ ID NO: 6 or 24; or

[0140] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 6 or 24, preferably, the amino acid alterations do not occur in the CDR region.

[0141] In some embodiments, the three complementarity-determining regions (HCDRs) of the anti-TROP2 antibody or its antigen-binding fragment described in this invention, HCDR1, HCDR2, and HCDR3, are selected from the heavy chain variable region.

[0142] (i) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1 or 23;

[0143] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0144] The HCDR can be determined according to any scheme for determining the CDR, such as kabat, AbM, Chothia, Contact, or IMGT or a combination thereof.

[0145] For example, the HCDR1 sequence is defined according to the Kabat and Chothia schemes, and the HCDR2 and HCDR3 sequences are defined according to the Kabat schemes, respectively.

[0146] In some embodiments, the three complementarity-determining regions (LCDRs) of the anti-TROP2 antibody or its antigen-binding fragment described in this invention, LCDR1, LCDR2, and LCDR3, are selected from the variable region of the light chain.

[0147] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:6 or 24, or

[0148] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0149] The LCDR1, 2 and 3 can be determined according to any scheme for determining CDR, such as kabat, AbM, Chothia, Contact or IMGT or a combination thereof.

[0150] For example, LCDR1, 2 and 3 are determined according to the Kabat scheme.

[0151] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises:

[0152] (i) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1 or 23, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:6 or 24.

[0153] (ii) the three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:1, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:6; or

[0154] (iii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:23, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:24.

[0155] The HCDR1, 2 and 3 and LCDR1, 2 and 3 can be determined according to any scheme for determining CDR, such as the schemes of kabat, AbM, Chothia, Contact or IMGT or combinations thereof.

[0156] For example, HCDR1 is determined according to the Kabat and Chothia schemes, and HCDR2 and HCDR3 are determined according to the Kabat scheme, and LCDR1, 2 and 3 are determined according to the Kabat scheme.

[0157] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:2, or HCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

[0158] In some embodiments, HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:3, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3.

[0159] In some embodiments, HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:4, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4.

[0160] In some embodiments, LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:7, or LCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

[0161] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:8, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0162] In some embodiments, LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:9, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

[0163] In some specific embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3). HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0164] In some embodiments, in the anti-TROP2 antibody or its antigen-binding fragment of the present invention, the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively comprise or are composed of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0165] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0166] (i) a VH comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:6 or a VL comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or

[0167] (ii) Contains the amino acid sequence shown in SEQ ID NO:23 or has at least 90% of it,

[0168] Amino acid sequences or VHs consisting of said amino acid sequences having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:24, and / or containing amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with said amino acid sequences.

[0169] Or an amino acid sequence with 99% identity or a VL composed of said amino acid sequence.

[0170] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment described in this invention comprises a heavy chain variable region and a light chain variable region, wherein

[0171] (i) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; or

[0172] (ii) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0173] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain. In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention further comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain of the present invention comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region. In some embodiments, the anti-TROP2 antibody of the present invention comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains. In some embodiments, the anti-TROP2 antibody of the present invention comprises two identical heavy chains and two identical light chains, or is composed of the latter.

[0174] In some implementations, the heavy chain includes

[0175] (i) Containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 5 or 58.

[0176] The sequence may be composed of or consist of the amino acid sequence described above;

[0177] (ii) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or

[0178] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 58.

[0179] In some implementations, the light chain comprises

[0180] (i) Containing amino acids having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 10 or 59.

[0181] The amino acid sequence or is composed of the amino acid sequence;

[0182] (ii) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or

[0183] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 59.

[0184] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment described in this invention comprises a heavy chain and a light chain, wherein

[0185] (i) the heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10; or

[0186] (ii) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:58, and the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:59.

[0187] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment described in this invention comprises a heavy chain and a light chain, wherein

[0188] (i) the heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:5, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:10; or

[0189] (ii) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:58, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:59.

[0190] In some embodiments, the anti-TROP2 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0191] (i) Showing the same or similar binding affinity and / or specificity to TROP2 as the anti-TROP2 antibody of the present invention (e.g., the chAb1 antibody or hzAb1.4 antibody in the examples);

[0192] (ii) Inhibit (e.g., competitively inhibit) the binding of the anti-TROP2 antibody of the present invention (e.g., the chAb1 antibody or hzAb1.4 antibody in the examples) to TROP2;

[0193] (iii) The same or overlapping epitopes that bind to the anti-TROP2 antibody of the present invention (e.g., the chAb1 antibody or hzAb1.4 antibody in the examples);

[0194] (iv) Competing with the anti-TROP2 antibody of the present invention (e.g., the chAb1 antibody or hzAb1.4 antibody in the examples) to bind to TROP2;

[0195] (v) Having one or more biological characteristics of the anti-TROP2 antibody of the present invention (e.g., the chAb1 antibody or hzAb1.4 antibody in the examples).

[0196] In some embodiments, the anti-TROP2 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, preferably an antibody in the form of IgG1.

[0197] In some implementations, the anti-TROP2 antibody is a monoclonal antibody.

[0198] In some embodiments, the anti-TROP2 antibody is humanized. In some embodiments, the humanized anti-TROP2 antibody of the present invention can be obtained by a method including the following steps:

[0199] ① Determine the CDR loop structure of the heavy chain variable region and light chain variable region of the parental antibody (e.g., a murine antibody selected from a hybridoma);

[0200] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the phylogenetic sequence database;

[0201] ③ Screen for the most closely matched human lineages and the lowest possible amount of reversion mutations to the heavy and light chains;

[0202] ④ Construct the CDR region of the chimeric antibody onto the human backbone region;

[0203] ⑤ Use sequence and structural features to determine the amino acid positions in the backbone region that maintain CDR function;

[0204] ⑥ Perform a reverse mutation (reverting to the input amino acid type) at the identified important sequence positions;

[0205] ⑦ Optimize amino acids at risk sites.

[0206] In some implementations, the anti-TROP2 antibody is a chimeric antibody.

[0207] In some embodiments, the anti-TROP2 antibody of the present invention is a full-length antibody.

[0208] In one embodiment, the anti-TROP2 antibody of the present invention also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies, or linear antibodies.

[0209] In one embodiment, the anti-TROP2 antibody of the present invention may also be a bispecific antibody or a multispecific antibody.

[0210] II-2 anti-PD-L1 antibody or its antigen-binding fragment

[0211] This invention provides a PD-L1 antibody with a strong binding affinity for PD-L1. In some embodiments, the PD-L1 antibody of this invention is suitable for constructing the antigen-binding region in a multispecific binding molecule.

[0212] In some embodiments, the anti-PD-L1 antibody of the present invention is capable of binding with high affinity to human PD-L1 and / or cynomolgus monkey PD-L1, for example, its K... D The value is less than or equal to about 20 nM, for example, less than or equal to about 15, 10, 9, 8, 7, 6, or 5 nM. In some embodiments, the anti-PD-L1 antibody of the present invention binds to human PD-L1 and / or cynomolgus monkey PD-L1. D Values ​​greater than approximately 0.5 nM, 1 nM, 2 nM, 3 nM, or 4 nM. In some embodiments, the anti-PD-L1 antibody of the present invention binds to human PD-L1 and / or cynomolgus monkey PD-L1. D The value falls between any two of the above-mentioned numerical ranges. In some embodiments, the binding affinity of the anti-PD-L1 antibody of the present invention is determined by thin-layer interferometry.

[0213] In some embodiments, the anti-PD-L1 antibody of the present invention can inhibit PD-L1 activity, for example, block PD-L1 / PD-1 activity.

[0214] In some embodiments, the anti-PD-L1 antibody of the present invention or its antigen-binding fragment can effectively bind to PD-L1, such as human or cynomolgus monkey PD-L1.

[0215] In some embodiments, the anti-PD-L1 antibody of the present invention or its antigen-binding fragment is internalized on PD-L1 positive cells, such as PD-L1 positive tumor cells, for example, with good internalization effect.

[0216] In some embodiments, the anti-PD-L1 antibody of the present invention also encompasses multispecific antibodies, such as bispecific antibodies, that specifically bind to PD-L1 and other antigens.

[0217] In some embodiments, the anti-PD-L1 antibody of the present invention is a single-domain antibody, such as a VHH antibody.

[0218] In some embodiments, the anti-PD-L1 single-domain antibody of the present invention is a VHH antibody comprising or composed of a heavy chain variable region, wherein the heavy chain variable region typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme described in the "Definitions" section, for example, the boundaries of the three CDRs in the VHH sequence can be defined by schemes such as Kabat, Chothia, AbM, or IMGT or combinations thereof.

[0219] In some embodiments, the anti-PD-L1 VHH antibody of the present invention comprises

[0220] (i) The three complementary determining regions (HCDR or VHH CDR) contained in the VH shown in SEQ ID NO: 25 or 31, or

[0221] (ii) The sequence relative to (i) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alteration (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions.

[0222] The HCDR can be determined according to any scheme for determining the CDR, such as kabat, AbM, Chothia, Contact, or IMGT or a combination thereof.

[0223] For example, the HCDR1 sequence is defined according to the Kabat and Chothia schemes, and the HCDR2 and HCDR3 sequences are defined according to the Kabat schemes, respectively.

[0224] In some embodiments, the anti-PD-L1 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising

[0225] (i) The three complementary determining regions (HCDR or VHH CDR) contained in VH as shown in SEQ ID NO:25 or 31, or

[0226] (ii) The sequence relative to (i) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alteration (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions.

[0227] The HCDR can be determined according to any scheme for determining the CDR, such as kabat, AbM, Chothia, Contact, or IMGT or a combination thereof.

[0228] For example, the HCDR1 sequence is defined according to the Kabat and Chothia schemes, and the HCDR2 and HCDR3 sequences are defined according to the Kabat schemes, respectively.

[0229] In some embodiments, the anti-PD-L1 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-PD-L1 VHH of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0230] In some embodiments, the VHH CDR1 described herein comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:26, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO:26.

[0231] In some embodiments, the VHH CDR2 described herein comprises or is composed of the amino acid sequence of SEQ ID NO:27, or the VHH CDR2 described herein comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:27.

[0232] In some embodiments, the VHH CDR3 described herein comprises or is composed of an amino acid sequence selected from SEQ ID NO:28, or the VHH CDR3 described herein comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO:28.

[0233] In one embodiment, the anti-PD-L1 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHHCDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:26, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:27, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:28.

[0234] In one embodiment, the anti-PD-L1 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHHCDR1, VHHCDR2, and VHHCDR3, wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:26, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:27, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:28.

[0235] In some embodiments, the anti-PD-L1 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region

[0236] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 25 or 31; or

[0237] (ii) Contains or consists of an amino acid sequence selected from or represented by SEQ ID NO: 25 or 31; or

[0238] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:25 or 31, preferably, the amino acid changes do not occur in the CDR region.

[0239] In some embodiments, the anti-PD-L1 VHH antibody of the present invention comprises or is composed of an amino acid sequence selected from or represented by SEQ ID NO:25 or 31.

[0240] In one embodiment, the anti-PD-L1 VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, particularly the framework region sequence, in a non-human natural VHH sequence (e.g., a VHH sequence derived from camel or alpaca immunization) with residues at the corresponding positions of the heavy chain VH from a conventional human antibody. Methods for humanizing VHH are well known in the art, such as those described in Example 3. Typically, humanization substitutions are performed in a manner that preserves the favorable binding properties of the single-domain antibody. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art, in order to identify and select suitable mutations or combinations of mutations in the humanized residues.

[0241] In some embodiments, the humanized single-domain antibody of the present invention can be obtained by a method including the following steps: ① determining the CDR loop structure of the parental single-domain antibody (e.g., a camel-derived VHH antibody screened from a phage display library);

[0242] ② Find the closest homologous sequence for each V / J region of VHH in the phylogenetic sequence database;

[0243] ③ Construct the CDR region of VHH onto the human skeletal region;

[0244] ④ Use sequence and structural features to determine the amino acid positions in the backbone region that maintain CDR function;

[0245] ⑤ Perform reverse mutations at sequence positions identified as important;

[0246] ⑥ Optimize amino acids at risk sites.

[0247] In another aspect of the invention, a heavy chain antibody comprising the variable region of the VHH antibody against PD-L1 of the present invention is also provided. In some embodiments, the single-domain antibody or VHH of the present invention (e.g., camel-derived VHH or its humanized form) may be linked to a constant region of a human antibody or a portion thereof, such as the Fc region, to produce a heavy chain antibody comprising the VHH-constant region, VHH-CH1-Fc, or VHH-Fc.

[0248] In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus. In some embodiments, VHH and Fc are linked by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., the hinge region of IgG1, 2, 3, or 4) or a portion thereof. In some embodiments, VHH and Fc are linked by a linker. In some embodiments, the linker comprises (GGGGS)n, where n = any integer between 1 and 5, such as 1, 2, 3, 4, or 5. In some embodiments, the linker comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:29.

[0249] In one aspect of the invention, the anti-PD-L1 antibody of the present invention is an anti-PD-L1 heavy chain antibody. In some embodiments, the anti-PD-L1 heavy chain antibody of the present invention comprises a heavy chain variable region as defined herein as anti-PD-L1 VHH or therein, and a heavy chain constant region or an Fc region of the heavy chain constant region, optionally linked by a linker or hinge region.

[0250] In some embodiments, the heavy chain antibody comprises a constant region derived from human or non-human primate (e.g., cynomolgus monkey) antibodies, such as a constant region derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0251] In some embodiments, the heavy chain antibody comprises an Fc region derived from a human or non-human primate (e.g., a cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises an Fc region derived from or derived from a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 Fc region.

[0252] In one embodiment, the heavy chain antibody according to the invention can dimerize with another polypeptide chain (e.g., another heavy chain antibody, the same or different) containing the Fc region via the Fc region. Therefore, in one embodiment, the invention also provides homologous or heteromeric proteins comprising the heavy chain antibody of the invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains or is composed of said two identical heavy chain antibody chains. Therefore, in some embodiments, the reference to "heavy chain antibody" also encompasses a dimer composed of two heavy chain antibody chains.

[0253] In some embodiments, the anti-PD-L1 antibody of the present invention comprises a heavy chain, the heavy chain comprising a heavy chain variable region and an Fc region (optionally connected by a hinge region or a connector). In some embodiments, the anti-PD-L1 antibody of the present invention comprises or is composed of a heavy chain, the heavy chain comprising or composed of the heavy chain variable region and Fc region of the anti-PD-L1 VHH of the present invention, optionally the variable region and Fc region being connected by a hinge region or a connector.

[0254] In some embodiments, the anti-PD-L1 heavy chain antibody of the present invention comprises or is composed of a heavy chain, said heavy chain

[0255] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 30 or 32; or

[0256] (ii) Contains or consists of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO: 30 or 32; or

[0257] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 30 or 32, preferably, the amino acid changes do not occur in the CDR region.

[0258] II-3 Multispecific Antibody

[0259] In one aspect of the invention, the invention relates to a multispecific binding molecule capable of specifically binding TROP2, as well as specifically binding to one or more other targets or antigens. In some embodiments, the other antigen is PD-L1.

[0260] In one aspect, the present invention relates to a multispecific binding molecule capable of specifically binding to PD-L1, and specifically binding to one or more other targets or antigens. In some embodiments, the other antigen is TROP2.

[0261] In some embodiments, the multispecific binding molecule is a multispecific antibody, such as a bispecific antibody.

[0262] In some embodiments, the antibody of the present invention is a bispecific antibody, such as an anti-TROP2×PDL1 bispecific antibody.

[0263] In some embodiments, the anti-TROP2×PD-L1 antibody of the present invention is capable of binding with high affinity to human TROP2, cynomolgus monkey TROP2, human PD-L1, and / or cynomolgus monkey PD-L1. For example, the bispecific antibody uses a K+ concentration of less than 20 nM. D Values ​​specifically bind to human and / or cynomolgus monkey PD-L1, and at K values ​​less than approximately 5, 4, 3, 2, 1.9, 1.8, 1.7, or 1.6 nM. D The value specifically binds to TROP2 in humans and / or cynomolgus monkeys. In some embodiments, the binding affinity of the present invention is determined by thin-layer interferometry of biological membranes.

[0264] In some embodiments, the anti-TROP2×PD-L1 antibody of the present invention can inhibit PD-L1 activity, for example, block PD-L1 / PD-1 activity.

[0265] In some embodiments, the anti-TROP2×PD-L1 antibody of the present invention is internalized on TROP2-positive and / or PD-L1-positive cells, such as TROP2-positive and / or PD-L1-positive tumor cells, for example, having a good internalization effect, for example, mediating more toxin molecules into tumor cells to produce a good tumor killing effect.

[0266] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0267] The first antigen-binding region and the second antigen-binding region specifically bind TROP2, and the second antigen-binding region specifically binds PD-L1.

[0268] The first antigen-binding region of the bispecific antibody applicable to the present invention may contain or be composed of the anti-TROP2 antibody of the present invention or its antigen-binding fragment, as long as it can specifically bind TROP2, including but not limited to, for example, full-length antibodies, haptens, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies or linear antibodies that specifically bind TROP2.

[0269] In some embodiments, the first antigen-binding region is derived from the anti-TROP2 antibody described herein or its antigen-binding fragment, such as the Fab of anti-TROP2. In some embodiments, the first antigen-binding region specifically binding to TROP2 comprises 1, 2, 3, 4, 5, or 6 CDRs of the anti-TROP2 antibody described herein. In some embodiments, the first antigen-binding region comprises 1, 2, and 3 heavy chain variable region CDRs of the anti-TROP2 antibody described herein, namely HCDR1, HCDR2, and HCDR3. In some embodiments, the first antigen-binding region comprises 1, 2, and 3 light chain variable region CDRs of the anti-TROP2 antibody described herein, namely LCDR1, LCDR2, and LCDR3. In some embodiments, the first antigen-binding region comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of the anti-TROP2 antibody described herein. In some embodiments, the first antigen-binding region comprises the heavy chain variable region of the anti-TROP2 antibody described herein. In some embodiments, the first antigen-binding region comprises the light chain variable region of the anti-TROP2 antibody described herein. In some embodiments, the first antigen-binding region comprises the heavy chain variable region and the light chain variable region of the anti-TROP2 antibody described herein. In some embodiments, the first antigen-binding region comprises or is composed of the Fab of the anti-TROP2 antibody described herein.

[0270] The Fab fragment suitable as the antigen-binding region of a bispecific antibody consists of two polypeptide chains comprising antibody VH, CH1, VL, and CL domains, wherein VH pairs with VL and CH1 pairs with CL to form the antigen-binding region. In some embodiments, in the Fab, one chain comprises VH and CH1 from the N-terminus to the C-terminus (i.e., VH-CH1) or consists of VH and CH1, and the other chain comprises VL and CL from the N-terminus to the C-terminus (i.e., VL-CL) or consists of VL and CL. In some embodiments, in the multispecific antibody of the present invention, the Fab can be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain containing VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and can be linked to the antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the link is a direct link or a linker link. In this article, Fab chains containing VH-CH1 or VH-CH1 are also called Fab heavy chains, while Fab chains containing VL-CL or composed of VL-CL are also called Fab light chains.

[0271] In some embodiments, the Fab fragment is derived from an anti-TROP2 antibody as defined in this invention, comprising the heavy chain variable region VH and the light chain variable region VL of the anti-TROP2 antibody as defined in this invention. In some embodiments, the Fab heavy chain in the Fab that specifically binds to TROP2 as the first antigen-binding region comprises VH and CH1, or is composed of VH and CH1, wherein VH is the VH of the anti-TROP2 antibody as defined in this invention. In some embodiments, the Fab light chain in the Fab that specifically binds to TROP2 as the first antigen-binding region comprises VL and CL, or is composed of VL and CL, wherein VL is the VL of the anti-TROP2 antibody as defined in this invention. In some embodiments, the first antigen-binding region comprises CH1 from IgG1 and / or the Kappa light chain constant region.

[0272] The second antigen-binding region of the bispecific antibody applicable to the present invention may contain an antibody against PD-L1 or its antigen-binding fragment, or be composed of such an antibody, as long as it can specifically bind to PD-L1, including but not limited to, full-length antibodies, haptens, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies that specifically bind to PD-L1.

[0273] In some embodiments, the second antigen-binding region is derived from the anti-PD-L1 antibody or its antigen-binding fragment described herein, such as a VHH antibody against PD-L1. In some embodiments, the second antigen-binding region of the bispecific antibody suitable for use in this invention may comprise the anti-PD-L1 VHH antibody described herein.

[0274] In some embodiments, the bispecific antibody of the present invention comprises an Fc dimer.

[0275] In one embodiment, the bispecific antibody may include one or more first antigen-binding regions. In one embodiment, the bispecific antibody may include one or more second antigen-binding regions. In one embodiment, the bispecific antibody includes two first antigen-binding regions and two second antigen-binding regions.

[0276] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region is the Fab fragment of the anti-TROP2 antibody as defined in the present invention, and the second antigen-binding region is the VHH antibody against PD-L1 as defined in the present invention.

[0277] In one embodiment, the bispecific antibody of the present invention comprises a heavy chain and a light chain, wherein...

[0278] The heavy chain from the N-terminus to the C-terminus comprises or consists of the following: the Fab heavy chain of the first antigen-binding region - the Fc region - the second antigen-binding region (VHH);

[0279] The light chain from the N-terminus to the C-terminus includes or consists of the following: the Fab light chain of the first antigen-binding region;

[0280] Optionally, the Fc region is connected to the second antigen-binding region (VHH) via a connector.

[0281] In some embodiments, the Fab heavy chain comprises or consists of a Fab heavy chain variable region and a heavy chain constant region CH1. In some embodiments, the Fab light chain comprises or consists of a Fab light chain variable region and a light chain constant region. In a specific embodiment, the first antigen-binding region Fab is the anti-TROP2 Fab as defined in this invention, and the second antigen-binding region VHH is the anti-PD-L1 VHH as defined in this invention.

[0282] In some implementations, the antigen-binding region, Fc region, and linker in the bispecific antibody are as defined herein.

[0283] In some embodiments, the linker is a linker composed of glycine, such as (G)n, where n = any integer between 5 and 15, for example, n = 10 or 11. In some embodiments, the linker comprises or is composed of the amino acid sequence shown in SEQ ID NO:51.

[0284] In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation, and / or the absence of a C-terminal lysine.

[0285] In some specific embodiments, the heavy chain of the bispecific antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0286] In some specific embodiments, the light chain of the bispecific antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0287] In some specific implementations, the bispecific antibody that specifically binds to TROP2 and PD-L1 comprises a heavy chain and a light chain, wherein

[0288] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or the light chain comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0289] In some specific implementations, the bispecific antibody that specifically binds to TROP2 and PD-L1 comprises a heavy chain and a light chain, wherein

[0290] The heavy chain contains or consists of the amino acid sequence shown in SEQ ID NO:49, and the light chain contains or consists of the amino acid sequence shown in SEQ ID NO:50.

[0291] In some embodiments, the bispecific antibody of the present invention comprises two heavy chains and two light chains, for example, two identical heavy chains and two identical light chains, or is composed of said heavy chains and light chains.

[0292] II-4 applies to the constant region or Fc region of the molecule of this invention.

[0293] In some embodiments, the antibody heavy chain constant region of the present invention suitable for molecules of the present invention, such as anti-TROP2 antibody or its antigen-binding fragment, anti-PD-L1 heavy chain antibody or multispecific antibody of the present invention, is derived from or is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, for example, derived from or is a constant region of human IgG1, IgG2, IgG3 or IgG4.

[0294] In some implementations, the heavy chain constant region

[0295] (i) Contains or is composed of an amino acid sequence selected from SEQ ID NO:21; or

[0296] (ii) An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:21.

[0297] In some embodiments, the antibody heavy chain constant region comprises or consists of CH1 and Fc regions, optionally connected by a hinge region (e.g., in the case where the CH1 or Fc region does not have a hinge region).

[0298] In some embodiments, the constant region of the antibody heavy chain of the present invention comprises an Fc region. Hereinafter, an Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include both native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass a wide range of naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present invention comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at its N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region, such as sequences D221 to P230 according to EU numbers. Mutations may be contained in said hinge region. Unless otherwise noted herein, the amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0299] In some embodiments, the Fc region of the antibody suitable for use in this invention is derived from or derived from human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises the amino acid sequence SEQ ID NO:54 or an amino acid sequence having at least 90% identity with said amino acid sequence, such as 95%, 96%, 97%, 98%, 99%, or higher, or is composed of said amino acid sequence.

[0300] In some embodiments, the constant region is modified within the Fc region. In one embodiment, the Fc region is modified to enhance the effector function (e.g., complement activation function) of the Fc region. In one embodiment, the effector function has been reduced or eliminated relative to the wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from: using a naturally occurring Fc isotype with reduced or eliminated effector function, and Fc region modification. The Fc region may also contain modifications that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor. In some embodiments, the Fc region used in the present invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor.

[0301] In some embodiments, the Fc region of the present invention contains mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation. In some embodiments, the Fc region...

[0302] (i) Contains or consists of an amino acid sequence selected from SEQ ID NO:55;

[0303] (ii) Contains an amino acid sequence with at least 85%, 90%, and

[0304] Amino acid sequences or sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity;

[0305] (iii) Contains an amino acid sequence with at least 85% or 90% of the amino acid sequence of SEQ ID NO:55.

[0306] Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and with L234A / L235A mutations.

[0307] In some embodiments, the heavy chain constant region of the present invention comprises a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation. In some embodiments, the heavy chain constant region...

[0308] (iv) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence;

[0309] (v) Contains an amino acid sequence with at least 85%, 90%, and [the amino acid sequence is similar to that of SEQ ID NO:56].

[0310] Amino acid sequences or sequences of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity;

[0311] (vi) Contains an amino acid sequence with at least 85%, 90%, and [the amino acid sequence is similar to that of SEQ ID NO:56].

[0312] Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and with L234A / L235A mutations.

[0313] In some embodiments, the heavy chain constant region or Fc region of the present invention lacks terminal lysine residues.

[0314] In some embodiments, CH1 is or is derived from IgG1, IgG2, IgG3, or IgG4, preferably CH1 derived from IgG1. In some embodiments, CH1

[0315] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:57;

[0316] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:57; or

[0317] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:57.

[0318] In some embodiments, the antibody light chain constant region of the present invention is or is derived from the lambda or Kappa light chain constant region, preferably the Kappa light chain constant region, such as the human lambda or Kappa light chain constant region. In some embodiments, the light chain constant region...

[0319] (i) Contains an amino acid sequence with at least 85% and 90% of the amino acid sequence of SEQ ID NO:22.

[0320] Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or consisting of said amino acid sequences; or

[0321] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:22.

[0322] II-5 antibody amino acid alteration

[0323] In one embodiment of the invention, the amino acid alteration described herein includes the substitution, insertion, or deletion of amino acids. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0324] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region.

[0325] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the sugars attached to it can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucosylation motif to enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0326] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0327] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0328] II-6 polynucleotides, vectors, and host cells

[0329] In one aspect, the present invention provides a nucleic acid encoding any of the above antibody molecules or their antigen-binding fragments (e.g., anti-TROP2 antibody or its antigen-binding fragment, anti-PD-L1 VHH antibody, anti-PD-L1 heavy chain antibody or the multispecific antibody of the present invention).

[0330] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0331] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 5, 6, 10, 23-25, 30-32, 49, 50, 58 or 59, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1, 5, 6, 10, 23-25, 30-32, 49, 50, 58 or 59.

[0332] The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0333] In one aspect, the present invention also provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0334] In one aspect, the invention also provides a host cell comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as HEK 293 or 293F cells or 293FT cells or Expi293 cells or Expi293F cells). In yet another embodiment, the host cell is prokaryotic.

[0335] Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can also be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. Suitable mammalian host cell lines for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO, HEK293, 293F, 293FT, or Expi293 cell.

[0336] II-7. Production and purification of antibody molecules

[0337] In another aspect, the present invention provides a method for producing the antibody molecule of the present invention or an antigen-binding fragment thereof, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chain to produce the antibody molecule or an antigen-binding fragment thereof under conditions suitable for assembling the polypeptide chain into the molecule.

[0338] To facilitate production and purification, antibody molecules or their antigen-binding fragments may be fused with a secretory signal peptide at the N-terminus or C-terminus (e.g., C-terminus), and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0339] For recombinant production, a polynucleotide encoding a polypeptide chain of the antibody molecule of the present invention or its antigen-binding fragment can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art.

[0340] In one embodiment, each polynucleotide encoding the antibody molecule of the present invention or its antigen-binding fragment can be transfected into a different vector, optionally the vector containing a heavy chain constant region or a light chain constant region (e.g., nucleotides encoding a heavy chain variable region are transfected into a vector containing a heavy chain constant region, or nucleotides containing a light chain variable region are transfected into a vector containing a light chain constant region, so that the vector can express the full-length heavy chain or the full-length light chain; or nucleotides encoding a heavy chain are transfected into a vector to express the full-length heavy chain, and nucleotides encoding a light chain are transfected into a vector to express the full-length light chain, so that the vector can express the full-length heavy chain or the full-length light chain).

[0341] Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. In one embodiment, one or more vectors encoding multiple chains of an antibody are transfected into the same host cell, such that the antibody chains assemble into a complete antibody within the host cell.

[0342] A variety of techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0343] The molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0344] The purity of the antibody molecules or their antigen-binding fragments of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0345] II-8. Determination Method

[0346] The antibody molecules or their antigen-binding fragments provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. Examples of methods for determining the properties of the antibody molecules or their antigen-binding fragments or bispecific antibodies of the present invention are provided, such as thin-layer interferometry, endocytosis activity assays, and PD1-NFAT-Reporter assays.

[0347] III. Immunoconjugates

[0348] III-1 antibody-drug conjugate

[0349] In one aspect, the present invention provides an immunoconjugate comprising the molecules of the present invention (e.g., antibody molecules or antigen-binding fragments thereof) and one or more other substances, such as labels or other active ingredients.

[0350] In one aspect, the present invention provides an immunoconjugate comprising the molecule of the present invention (e.g., an antibody molecule or an antigen-binding fragment thereof) and one or more other active ingredients (e.g., active ingredients derived from a medicament or therapeutic agent for treating the disease of the present invention, such as small molecules that enhance the therapeutic effect of the molecule of the present invention).

[0351] In some implementations, the immune conjugate is an antibody-drug conjugate (ADC).

[0352] This invention provides an antibody-drug conjugate having formula (I):

[0353] Ab-(LD) p (I)

[0354] Or its pharmaceutically acceptable salts or solvates,

[0355] in:

[0356] Ab is an antibody or fragment thereof, such as an antigen-binding fragment, that binds to TROP2 and / or PD-L1 (e.g., human TROP2 and / or PD-L1).

[0357] L is the connector;

[0358] D represents a drug, preferably an anti-tumor compound; and

[0359] p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0360] In some embodiments, the Ab suitable for the ADC molecule of the present invention may comprise or consist of the antibody molecule of the present invention or its antigen-binding fragment, as long as it can specifically bind TROP2 and / or PD-L1, including but not limited to, full-length antibodies, haptens, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies that specifically bind TROP2 and / or PD-L1.

[0361] In some embodiments, the Ab suitable for the ADC molecule of the present invention may be the anti-TROP2 antibody of the present invention or its antigen-binding fragment, or the antibody of the present invention that specifically binds to PD-L1, or the multispecific antibody of the present invention that specifically binds to TROP2 and PD-L1.

[0362] In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and contains substituents or partial structures that can be attached to the linker structure; there are no particular limitations. For example, the antitumor compound can be a pharmaceutically active compound that acts on tumors. For antitumor compounds, it is preferable that part or all of the linker can be cleaved within tumor cells, releasing the antitumor compound portion, thereby exhibiting an antitumor effect. When the linker is cleaved at the linker portion with the drug, the antitumor compound is released in its unmodified structure, thus exerting its original antitumor effect.

[0363] In some implementations, the antitumor compound may be, for example, a cytotoxic agent or a chemotherapeutic agent, such as camptothecin compounds such as eczema, DXd, auristatin compounds such as monomethyl auristatin E (MMAE), MMAF, or maytansine compounds such as the small molecule microtubule inhibitor DM1.

[0364] In some implementations, D has the formula -QL 2 -L 1 -D 1 The structure,

[0365] Where Q is either -O- or -S-;

[0366] L 1 Is it non-existent or -(C1-C)? 10 alkylene);

[0367] L 2 It does not exist, *-(C1-C 10 alkylene)-C(O)N(R 5 )- or *-(C1-C 10 Alkane

[0368] (base)-N(R) 5 )C(O)-; where * indicates that the end is covalently connected to Q; and R 5 It is an H or C1-C6 alkyl group.

[0369] Where D 1 It has the structure shown in equation (D-1):

[0370]

[0371] Where R 1Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0372] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and -SR 4 ;R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 Or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0373] R 4 Selected from H or C1-C4 alkyl groups.

[0374] In some implementation schemes, R 1 For H, R 2 It is a C1-C6 alkyl or C1-C6 alkoxy, R 3 For halogens, -F is preferred.

[0375] In some implementation schemes, D 1 It has the structure shown in equation (D-2):

[0376]

[0377] Where R 1 R 2 and R 3 As defined above.

[0378] In some implementation schemes, D 1 It has the structure shown in equation (D-3):

[0379]

[0380] In some implementation schemes, D 1 It has the structure shown in equation (D-4):

[0381]

[0382] In some implementations, -L 2 -L 1 - is -(C1-C6 alkylene)-, *-(C1-C6 alkylene)

[0383] (base)-C(O)N(R) 5)-(C1-C6 alkylene)- or *-(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-, where * indicates that the terminal is covalently connected to Q;

[0384] R 5 It is H or C1-C6 alkyl.

[0385] In some implementations, -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0386] In some implementations, -QL 2 -L 1 The symbol is -OCH2-CH2-CH2-CH2-. It should be understood that the left side of this group is connected to the L phase.

[0387] In some implementations, -L- has the following structure: -ZE-NH-CH2-

[0388] Where Z is connected to Ab, and -CH2- is connected to Q;

[0389] Z is selected from

[0390]

[0391] Where m a1 and m a2 Integers independently selected from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;

[0392] m is selected from an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, or 8.

[0393] Preferred Where m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5; the carbonyl group on its right end is covalently connected to E;

[0394] E is a peptide residue containing 2-10 amino acids, wherein the peptide residue is optionally substituted by one or more (e.g., 2, 3, or 4) groups selected from C. 1-6 Alkyl and polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z.

[0395] In some implementations, E is a peptide residue containing 2, 3, or 4 amino acids.

[0396] In some embodiments, the amino acid is selected from, for example, glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution. In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution.

[0397] In some embodiments, the substituted glutamine or glutamic acid has the structure shown below: Where R 6 It is an H or C1-C6 alkyl group;

[0398] Preferred

[0399] Where R 6 It is H or C1-C6 alkyl.

[0400] In some implementations, E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu-Cit-, -Lys-Cit -, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-Glu-, -Val-L ys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-L ys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys-Leu-, -A la-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution,

[0401] Preferably, the substituted Gln or Glu has the structure shown in formula (G-1a), formula (G-1a), or formula (G-1a) as described above.

[0402] In some implementations, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or Where R 6 It is an H or C1-C6 alkyl group, wherein these E groups are covalently linked to Z via the left-hand N-terminus.

[0403] In some implementations, -ZE-NH-CH2- has the following structure

[0404]

[0405] It should be understood that the left end of this structure is connected to the Ab part.

[0406] In some embodiments, the payload (e.g., a drug) of the immunoconjugate of the present invention is attached to the modified antibody or antibody fragment via a thiol group of a free cysteine ​​residue (optionally via a linker). In some embodiments, the payload (e.g., a drug) of the immunoconjugate of the present invention is attached to the thiol group of the cysteine ​​residue via a cleavable or non-cleavable linker.

[0407] In some implementations, Ab is linked to L via a sulfur atom on its thiol group (e.g., a thiol group generated by disulfide bond reduction), in which case the antibody-drug conjugate of formula (I) can be represented by the following formula:

[0408] Ab'-(SLD) p (I')

[0409] Ab' is defined as Ab above, and L, D, and p are defined as above.

[0410] In some embodiments, the antibody-drug conjugate has an average DAR value of 2-6, 2-5, 3-5, or approximately 3.5, 3.6, 3.7, 3.9, or 4.

[0411] In some embodiments, the antibody-drug conjugate is selected from...

[0412]

[0413] Wherein Ab is a bispecific antibody of the present invention that specifically binds TROP2 and PD-L1; q is as defined above for p, preferably, the antibody-drug conjugate has an average DAR value of 2-6, 2-5, 3-5 or about 3.5, 3.6, 3.7, 3.9 or 4.

[0414] It should be understood that the S atom linked to Ab in the above ADC comes from the antibody Ab. The Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a thiol group -SH, which is then linked to the maleimide portion of the linker.

[0415] III-2. Preparation of the ADC molecule of the present invention

[0416] Another aspect of the present invention provides a method for preparing an antibody-drug conjugate (ADC) using the antibody of the present invention. In this invention, "ADC" is defined as an antibody coupled to an active substance (D) having biological and / or pharmaceutical activity via a linker (L). The method comprises coupling an antibody (Ab) of the present invention to one or more active substances D via one or more linkers (L) defined in the present invention.

[0417] In some implementations, the method includes the following steps:

[0418] (a) Add antibody Ab to buffer solution, add reducing agent, and then incubate;

[0419] (b) Add a linker-loaded polymer to the reaction solution from step (a) for coupling to obtain the crude product; and

[0420] (c) Optionally, the crude product is purified to obtain the antibody-drug conjugate of the present invention;

[0421] Ab is defined as above.

[0422] It should be understood that the linker-loaded compound reacts with Ab to provide the -LD portion in compound I, and the structure of the linker-loaded compound can be determined when -LD is clearly defined.

[0423] In some implementations, the buffer solution in step a) is a PBS buffer, preferably with a pH of 5.0-9.0, for example 6.0-8.0.

[0424] In some implementations, the reducing agent in step a) is TCEP.

[0425] In some implementations, the connector-payload has the following structure: Z'-E-NH-CH2-QL 2 -L 1 -D 1 Among them, E, Q, L 2 L 1 D 1 As defined above, Z' is the same as Z as defined above, except that the maleimide group in Z is removed. Replace with m is as defined above.

[0426] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments.

[0427] It should be noted that implementation schemes obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 80%, 60%, 40%, 20%, or 10% are also under consideration in this invention.

[0428] IV. Pharmaceutical Compositions

[0429] In some embodiments, the present invention provides compositions comprising molecules of the present invention (e.g., antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules) or their pharmaceutically acceptable salts, preferably pharmaceutical compositions or pharmaceutical formulations.

[0430] In one embodiment, the composition further comprises a pharmaceutical excipient. In some embodiments, the pharmaceutical excipient is, for example, a pharmaceutical carrier, a pharmaceutical excipient, or a buffer known in the art.

[0431] In one embodiment, the composition, for example, a pharmaceutical composition, comprises the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules), and a combination of one or more other therapeutic agents.

[0432] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0433] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0434] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0435] A pharmaceutical product comprising the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates, such as ADC molecules) can be prepared by mixing molecules of the present invention having the desired purity (e.g., the antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates, such as ADC molecules) with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0436] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably combined in amounts effective for the intended use.

[0437] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0438] V. Drug combinations and pillboxes

[0439] In some embodiments, the present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the molecules of the present invention (e.g., antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules), and one or more other therapeutic agents.

[0440] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0441] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0442] - A first container containing a pharmaceutical composition comprising molecules of the present invention (e.g., antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules);

[0443] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0444] In some embodiments, when the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules) are used to treat tumors, other therapeutic agents that may be combined with or used in combination with the molecules of the present invention cover a variety of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0445] Other exemplary antibodies include those that specifically bind to immune checkpoints.

[0446] VI. Uses and Methods

[0447] This invention provides, in one aspect, a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., an antibody molecule of the invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), a pharmaceutical composition, a drug combination, or a kit. In some embodiments, the disease is a TROP2 and / or PD-L1 related disease and / or condition. In some embodiments, the disease is, for example, an oncology such as cancer.

[0448] In some embodiments, the present invention relates to molecules of the invention (e.g., antibody molecules of the invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules), pharmaceutical compositions, pharmaceutical combinations or kits for activating the immune system.

[0449] In some embodiments, the present invention relates to molecules of the invention (e.g., antibody molecules of the invention or their antigen-binding fragments or their immunoconjugates such as ADC molecules), pharmaceutical compositions, pharmaceutical combinations or kits, for use in therapies, such as for treating TROP2 and / or PD-L1-related diseases and / or conditions.

[0450] In some embodiments, the present invention relates to methods of treating diseases, such as those mentioned herein, using molecules of the present invention (e.g., antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates, such as ADC molecules), pharmaceutical compositions, pharmaceutical combinations or kits, or uses for said treatment, or uses for preparing medicaments for said treatment.

[0451] In some embodiments, the disease is a TROP2 and / or PD-L1 related disease and / or condition. In some embodiments, the disease is, for example, an oncology such as cancer.

[0452] In some embodiments, the tumor is a solid tumor or a hematologic malignancy, as well as a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, intermediate, or late stage, or metastatic stage. In some embodiments, the tumor is immune-evading.

[0453] In some embodiments, the tumor is a TROP2 and / or PD-L1 positive tumor or cancer, preferably a TROP2 and PD-L1 positive tumor or cancer. In some embodiments, the cancer is selected from pancreatic cancer, esophageal cancer, non-small cell lung cancer, breast cancer, or colon cancer, etc.

[0454] In some embodiments, a TROP2 and / or PD-L1 positive tumor or cancer refers to abnormal expression or activity of TROP2 and / or PD-L1 in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has TROP2 and / or PD-L1 expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) TROP2 and / or PD-L1 (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) TROP2 and / or PD-L1 (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to TROP2 and / or PD-L1 in adjacent healthy tissue or cells of the subject.

[0455] In some embodiments, a TROP2 and / or PD-L1 positive tumor or cancer refers to tumor cells in an individual with any type of tumor or cancer that express TROP2 and / or PD-L1. In some embodiments, the tumor cells of the individual express TROP2 and / or PD-L1, for example, moderate or high expression of TROP2 and / or PD-L1. In some embodiments, a TROP2 and / or PD-L1 positive tumor refers to an abnormal expression of TROP2 and / or PD-L1 in tumor cells. In some embodiments, abnormal expression of TROP2 and / or PD-L1 refers to higher TROP2 and / or PD-L1 expression on tumor cells compared to TROP2 and / or PD-L1 expression in control cells (e.g., healthy cells in the corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells).

[0456] In some embodiments, the tumors or cancers suitable for prevention or treatment by applying the anti-TROP2 antibody or its-based immune conjugates of the present invention are TROP2-positive tumors or cancers.

[0457] In some embodiments, the tumors or cancers suitable for prevention or treatment by applying the anti-PD-L1 antibody or its immunoconjugate of the present invention are PD-L1 positive tumors or cancers.

[0458] In some embodiments, tumors or cancers suitable for prevention or treatment using the bispecific antibodies or immunoconjugates based thereon of the present invention are TROP2 and PD-L1 positive tumors or cancers. In a preferred embodiment, the TROP2 and PD-L1 positive tumors or cancers refer to tumors or cancers exhibiting high expression of TROP2 and high, intermediate, or low expression of PD-L1 in tumor cells.

[0459] In one specific embodiment, the molecules of the present invention, such as any antibody or its antigen-binding fragment or immune conjugate, such as ADC molecules, are capable of killing tumor cells and / or inhibiting tumor cell proliferation, such as tumor cells expressing TROP2 or PD-L1, preferably tumor cells expressing both TROP2 and PD-L1, and more preferably tumor cells that highly express TROP2 and highly, moderately or poorly express PD-L1.

[0460] In some implementations, the tumor is a tumor immune escape.

[0461] In some embodiments, the tumor is a tumor that has already been treated with other treatments such as chemotherapy and / or radiotherapy.

[0462] Depending on their therapeutic use, the molecules (e.g., antibody molecules or their antigen-binding fragments or their immunoconjugates, etc.) or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as other treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0463] In some embodiments, when the molecules of the present invention (e.g., antibody molecules or their antigen-binding fragments or their immune conjugates) are used to treat tumors, the treatment methods include surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0464] In other respects, the present invention provides the use of the molecules of the present invention (e.g., antibody molecules or their antigen-binding fragments or their immunoconjugates, etc.) or compositions or combinations thereof in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0465] In other respects, the present invention also provides molecules of the present invention (e.g., antibody molecules or their antigen-binding fragments or their immunoconjugates), or compositions or pharmaceuticals or formulations or combination products comprising the present invention, for use in therapies, such as for treating the related diseases or conditions mentioned herein.

[0466] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0467] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which the administration of the molecules of the present invention (e.g., antibody molecules or their antigen-binding fragments or their immunoconjugates, etc.) or compositions or pharmaceutical preparations containing them may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceutical agents.

[0468] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0469] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0470] In some embodiments, when the molecules of the present invention (e.g., the antibody molecules of the present invention or their antigen-binding fragments or their immunoconjugates, such as ADC molecules) are used to treat tumors, other therapeutic agents that may be combined with or administered in combination with the molecules of the present invention cover a wide range of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Other exemplary antibodies include antibodies that specifically bind to immune checkpoints.

[0471] In one aspect, the present invention also relates to methods for diagnosing and detecting antibodies of the present invention or antigen-binding fragments thereof, and compositions comprising such antibodies for diagnosing and detecting.

[0472] In some implementations, any antibody molecule or its antigen-binding fragment provided herein may be used to detect the presence of TROP2 and / or PD-L1 in biological samples.

[0473] In some implementations, any anti-TROP2 antibody or its antigen-binding fragment provided herein can be used to detect the presence of TROP2 in biological samples.

[0474] In some implementations, any anti-PD-L1 antibody or its antigen-binding fragment provided herein can be used to detect the presence of PD-L1 in biological samples.

[0475] In some implementations, any anti-TROP2×PD-L1 antibody provided herein can be used to detect the presence of TROP2 and / or PD-L1 in biological samples.

[0476] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues.

[0477] In one implementation, an antibody molecule or its antigen-binding fragment is provided for use in a diagnostic or detection method.

[0478] In another aspect, a method for detecting the presence of TROP2 and / or PD-L1 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of TROP2 and / or PD-L1 proteins in the biological sample. In some embodiments, TROP2 and / or PD-L1 is human TROP2 and / or PD-L1. In some embodiments, the method comprises contacting the biological sample with an antibody molecule or fragment thereof as described herein (e.g., when used for detecting TROP2, the anti-TROP2 antibody or its antigen-binding fragment or the anti-TROP2×PD-L1 antibody of the present invention may be used; when used for detecting PD-L1, the anti-PD-L1 antibody or its antigen-binding fragment or the anti-TROP2×PD-L1 antibody of the present invention may be used; when used for detecting both TROP2 and PD-L1, the anti-TROP2×PD-L1 antibody of the present invention may be used) under conditions that allow the antibody molecule or fragment thereof to bind to TROP2 and / or PD-L1, and detecting whether a complex is formed between the antibody molecule or fragment thereof and TROP2 and / or PD-L1. The formation of the complex indicates the presence of TROP2 and / or PD-L1. This method can be in vitro or in vivo. In one embodiment, an antibody molecule or a fragment thereof is used to select subjects suitable for treatment utilizing the antibody molecule or a fragment thereof, for example, where TROP2 and / or PD-L1 are biomarkers used for selecting said subjects.

[0479] In some embodiments, a labeled antibody molecule or fragment thereof is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0480] In some embodiments provided herein, the sample is obtained prior to treatment with the molecules of the invention (e.g., the antibody molecules of the invention or their antigen-binding fragments or their immunoconjugates, such as ADC molecules), pharmaceutical compositions, drug combinations, or kits. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0481] In some implementations, TROP2 and / or PD-L1 are detected before treatment, for example, before initiating treatment or before a treatment after a treatment interval.

[0482] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of TROP2 and / or PD-L1, thereby determining TROP2 and / or PD-L1 values; comparing the TROP2 and / or PD-L1 values ​​with control values; and if the TROP2 and / or PD-L1 values ​​are greater than the control values ​​(preferably both TROP2 and PD-L1 values ​​are greater than the control values), administering to the subject a therapeutically effective amount of a molecule of the present invention (e.g., an antibody molecule of the present invention or its antigen-binding fragment or its immunoconjugate, such as an ADC molecule), pharmaceutical composition, drug combination, or kit, optionally in combination with one or more other therapies, thereby treating the disease.

[0483] These and other aspects and embodiments of the invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0484] Example 1: Discovery and Activity Detection of Mouse-Derived Anti-TROP2 Antibodies

[0485] 1.1 TROP2 antigen immunization

[0486] Human TROP2 extracellular protein (Acro, Cat#TR2-H5223) was emulsified with TiterMax (sigma, Cat#T2684) and then immunized Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) four times, with subcutaneous injections every two weeks (50 μg protein per mouse).

[0487] 1.2 Cell fusion and high-throughput screening

[0488] Once the serum titer met the requirements, the spleen of the mouse was harvested to prepare a suspension of B lymphocytes, which was then electrofused with SP2 / 0 myeloma cells (ATCC). The fused cells were diluted to 1–2 × 10⁴ cells / ml with selective medium (1640 medium containing 20% ​​FBS and 1x HAT), and seeded into 96-well plates, with 100 μl of cell suspension added to each well. On day 7 post-fusion, the selection medium (1640 medium containing 10% FBS and 1x HT) was replaced. The supernatant was collected for analysis after day 10 (or longer, depending on cell growth status).

[0489] Hybridoma cells specifically expressing anti-TROP2 antibody were screened using flow cytometry (FACS). The cells to be tested (huTROP2 / GS-CHO & cynoTROP2 / GS-CHO, internally constructed) were counted and diluted to 1E6 cells / ml, with 100 μl added to each well of a U-bottom 96-well plate. The cells were centrifuged at 500g for 5 min to remove the cell culture medium. The supernatant from the hybridoma 96-well plate culture was added to a U-shaped plate to resuspend the cells, 100 μl per well, and incubated on ice for 30 min. The cells were then centrifuged at 500g for 5 min to remove the supernatant and washed once with PBS. Incubate at 500g for 5 min to remove PBS. Add 100 μl of FITC-labeled secondary antibody against mouse Fab (Jackson Immunoresear, Cat#115-545-006) (1:500 diluted in PBS) to each well. Add 100 μl of PE-labeled secondary antibody against human Fc (Biolegend, Cat#409304) to the positive control antibody. Incubate on ice in the dark for 30 min. Incubate at 500g for 5 min to remove supernatant. Wash cells once with PBS. Resuspend cells in 50 μl of 1×PBS and analyze using FACS. Perform affinity assays on cell-binding positive clones. Subclone the final clones using limiting dilution and pick single-clone cells.

[0490] 1.3 Subclones of positive hybridoma cells

[0491] Limiting dilution subcloning procedure: Prepare a 96-well plate and add 200 μL of culture medium to each well. This culture medium is the same as the selection medium, except that HAT is replaced with HT (Gibco, Cat#11067-030). Prepare a cell suspension from the positive wells selected by fusion screening. Add 100 μL of this suspension to each well in the first row and mix well. Then, add 100 μL of the cell suspension from the first row to the second row, mix thoroughly, and add 100 μL to the next row. Repeat the above steps, let the 96-well plate stand for 30 minutes, and observe and count the cells under a microscope. Take the volume corresponding to 100 cells, add 20 mL of culture medium, mix well, and plate the plate (200 μL per well). After one week, observe under a microscope, identify, and label the single-cloning wells.

[0492] When the cell confluence in each well reaches more than 50%, the high-throughput screening method described above is used to detect and select the target positive wells. After expansion culture, the cells are cryopreserved.

[0493] 1.4 Preparation of chimeric antibodies

[0494] The hybridoma candidate clones obtained in the above experiments were subjected to antibody light and heavy chain gene sequences, and constructed into a human-mouse chimeric antibody chAb1. The corresponding sequences of the antibody light and heavy chains are as follows: the constant region of the antibody heavy chain is the constant region of the human IgG1 heavy chain (SEQ ID NO:21), and the constant region of the light chain is SEQ ID NO:22. The variable regions and CDR sequences are shown in Table 1 below.

[0495] Freshly cultured cell lines, approximately 5E6 cells per line, were used to extract RNA (Macherey-Nagel, Cat#740984.250). cDNA was obtained through reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on the base sequence located in the 5' FR1 region, and downstream primers were designed based on the base sequence located in the antibody constant region or FR4 region to amplify the variable region gene fragments of the antibody light and heavy chains. These fragments were ligated into a T-vector (Mighty TA-cloning Kit, Takara), and single clones were selected for sequencing. The sequencing results were analyzed and compared using MEGA7 software.

[0496] Clones with correct and paired antibody light and heavy chain variable region sequences were subjected to homologous recombinase assays (PCR) by Novizan Pharmaceuticals in Nanjing. II, catalog number: C112-01) was ligated into the pcDNA3.1 vector (where the constant region of the IgG1 heavy chain is CH1CH2CH3 (SEQ ID NO:21) or the constant region of the Kappa light chain (SEQ ID NO:22)), wherein the constant region was selected from the IgG1 subtype, to obtain expression plasmids for light chain and heavy chain antibodies.

[0497] Table 1: CDR light and heavy chain sequence information for TROP2 chimeric antibody

[0498] chAb1 sequence hRS7 VH 1 11 CDRH1 2 12 CDRH2 3 13 CDRH3 4 14 VL: 6 16 CDRL1 7 17 CDRL2 8 18 CDRL3 9 19

[0499] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF#A14351-01). Cell density was checked one day before transfection (viability should be greater than 95%), and the cell density was adjusted to 3E6 cells / mL with fresh Expi293F medium and cultured for another day. On the day of transfection, the cell density was adjusted to 3E6 cells / mL.

[0500] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as transfection buffer, add the plasmid of the chimeric antibody to be transfected at a ratio of 1 mg / L, with the light chain plasmid ratio being 1:1, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 min, then gently pour the mixture into Expi293F cell suspension while shaking, place the cells in a shaker for culture under the following conditions: 8% CO2, 36.5℃, 120 rpm.

[0501] After culturing for 16–18 hours, 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), 5 g / L glucose solution, and 2.2 mM Valproic acid sodium salt (Merk, Cat#P4543-100G) were added to the cell suspension. The mixture was gently stirred and incubated for another 7 days at 36.5°C and 120 rpm with 8% CO2. The cells were then harvested. The cell suspension was mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0502] Affinity chromatography was used to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing column was washed with 10 column volumes of 1×PBS to remove non-specifically binding proteins. The packing column was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, filtered for sterilization, and used for subsequent functional analysis after the purity test was qualified.

[0503] Using the same method described above, prepare the light and heavy chain plasmids or control IgG1 (control IgG1 (heavy chain: SEQ ID NO:52; light chain: SEQ ID NO:53)) corresponding to hRS7 (WO2003 / 074566) in Table 1 and express the corresponding antibodies for subsequent experiments.

[0504] 1.5 Affinity detection of chimeric anti-TROP2 antibody chAb1

[0505] In this study, the binding kinetics (KD) of the antibody-antigen in this invention was determined using biomembrane thin-layer interferometry (BLI). The affinity determination by BLI was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

[0506] Half an hour before the experiment, according to the sample quantity, take an appropriate number of AHC sensors (18-5060, Sartorius) and soak them in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). Dilute the anti-TROP2 antibody and recombinant human TROP2 protein (Human Trop2, Acro, Cat#TR2-H5223) or recombinant rhesus monkey Trop2 protein (Rhesus Trop2, Acro, Cat#TR2-C52H5) to 100 nM respectively.

[0507] SD buffer, antibody solution, and TROP2 recombinant protein were added to 96-well black polystyrene microplates (Greiner, 655209), respectively. Detection was performed using a Fortebio Octet Red96e. The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: baseline equilibration 120s, antibody loading and solidification 100s, baseline equilibration 120s, antigen binding 100s, and dissociation 120s; rotation speed 1000 rpm; temperature 30℃. After the experiment, the KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in Table 2 below.

[0508] The kinetic parameters from the affinity experiment show that the chimeric TROP2 antibody chAb1 has a strong affinity for recombinant human TROP2 protein, which is stronger than that of the control antibody hRS7 (DOI:10.1021 / acs.bioconjchem.5b00223).

[0509] Table 2: Affinity of anti-TROP2 chimeric antibody chAb1 to TROP2

[0510]

[0511] 1.6 Detection of endocytic activity of chimeric anti-TROP2 antibody chAb1

[0512] Add 10% fetal bovine serum (SH30406.05, HYCLONE) and 1% penicillin-streptomycin antibiotics (15140-122, GIBCO) to EMEM (30-2003, ATCC) and mix well to prepare complete culture medium. Adjust the density of FaDu cells (ATCC, HTB-43) to 5E4 cells / ml using complete culture medium, and seed 50 μl per well in F96 MicroWell microplates (167008, NUNC). Seal the plates with 200 μl of sterile PBS and incubate overnight at 37°C.

[0513] The initial concentrations of IgG1, hRS7, and chAb1 antibodies were adjusted to 400 nM (4X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 8 concentrations, with the last concentration being 0 nM. Similarly, the initial concentration of Fabzap (IT-51, ATSBIO) was adjusted to 800 nM (4X) using complete medium, and then serially diluted 4-fold to a total of 10 concentrations, with the last concentration being 0 nM.

[0514] Mix an equal volume of the antibody to be tested with Fabzap (antibody:Fabzap = 1:2) and incubate at 37°C for 30 min. Add 50 μl of the incubated mixture to each well of the plated cells and incubate at 37°C for 3 days. Remove the plate from the incubator and allow it to equilibrate to room temperature. Remove the Luminescent Cell Viability Assay kit (DD1101-02, Vazyme) from the -20°C freezer and allow it to equilibrate to room temperature. Add 100 μl of the test solution from the kit to each well, protect from light, shake at 150 rpm for 2 min, let stand for 10 min, and then detect the luminescence value using a multi-functional microplate reader (SPARK, TECAN).

[0515] The cell viability at each concentration was calculated using the formula: Cell Viability (%) = (OD value - each concentration) / (OD value - 0 nM) × 100%. A three-parameter fitting was performed using GraphPad Prism 10, with the fitting formula being Y = Bottom + (Top - Bottom) / (1 + (X / IC50)).

[0516] like Figure 1 As shown, chAb1 exhibits excellent endocytosis activity in the Fadu cell line, with a stronger activity than the control antibody hRS7. The IC50 values ​​indicate that the endocytosis activity of chAb1 is more than twice that of hRS7.

[0517] Example 2: Humanization, preparation and activity detection of mouse anti-TROP2 antibody chAb1

[0518] The chimeric antibodies obtained from hybridomas are humanized using conventional methods, and the specific steps are as follows:

[0519] ① Determine the CDR ring structure;

[0520] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the phylogenetic sequence database;

[0521] ③ Screen for the most closely matched human lineages and the lowest possible amount of reversion mutations to the heavy and light chains;

[0522] ④ Construct the CDR region of the chimeric antibody onto the human backbone region;

[0523] ⑤ Use sequence and structural features to determine the amino acid positions in the backbone region that maintain CDR function;

[0524] ⑥ Perform a reverse mutation (reverting to the input amino acid type) at the identified important sequence positions;

[0525] ⑦ Optimize amino acids at risk sites.

[0526] The final humanized antibody, hzAb1.4, was thus obtained. Its CDR sequence, light chain variable region, and heavy chain variable region sequences, as well as the amino acid sequences of the light and heavy chains, are shown in the sequence listing and Table 3. The heavy chain constant region was selected from the human IgG1 sequence (SEQ ID NO:21), and the light chain constant region was selected from the corresponding constant region CL-Kappa (SEQ ID NO:22) based on the variable region being Kappa. The heavy and light chain sequences of the antibody were then constructed into the expression vector pcDNA3.1 (Invitrogen, V790-20), and transfected into Expi293 cells (Invitrogen, A14527) to obtain the corresponding humanized antibody. The specific transfection process was the same as in Example 1.

[0527] Table 3: Comparison of light and heavy chain combinations of TROP2 chimeric antibody chAb1 humanized CDR mutant antibody combinations

[0528]

[0529] Antibody preparation: Refer to the antibody preparation process in Example 1.

[0530] 2.1 Affinity detection of humanized anti-TROP2 antibody

[0531] The affinity of the humanized TROP2 antibody of the present invention was determined using the biofilm thin-layer interferometry (BLI) technique mentioned in Example 1. The affinity of the humanized molecule is shown in Table 4 below.

[0532] Table 4: Affinity test of anti-TROP2 antibody to TROP2 before and after humanization

[0533] Sample ID KD(M) kon(1 / Ms) kdis(1 / s) chAb1 6.46E-10 3.10E+05 2.00E-04 hzAb1.4 7.53E-10 2.66E+05 2.00E-04

[0534] The antibody affinity test results are shown in Table 4. The humanized antibody still has a high affinity for the antigen TROP2, and it has an equilibrium dissociation constant KD that is comparable to that of the corresponding chimeric antibody against the antigen.

[0535] 2.2 Detection of endocytic activity of humanized anti-TROP2 antibody

[0536] Following the method used in Example 1.6, we re-evaluated the endocytic activity of the humanized antibody hzAb1.4 on FaDu cells.

[0537] like Figure 2 As shown, hzAb1.4 still maintains good endocytosis in the Fadu cell line, and its activity is comparable to that of its parent antibody chAb1.

[0538] Example 3: Discovery and Activity Detection of Alpaca-Derived Anti-PD-L1 Antibodies

[0539] 3.1 PD-L1 antigen immunization and library preparation

[0540] Recombinant PD-L1 protein (ACRO, Cat#PD1-H 5258) was emulsified with Freund's adjuvant and used to subcutaneously immunize two healthy adult alpacas at multiple sites, with booster immunizations every 2-3 weeks for a total of five immunizations. Peripheral blood was collected from the immunized alpacas, lymphocytes were isolated, and total mRNA was extracted from PBMCs using Trizol reagent. cDNA was amplified using the PrimeScript reverse transcription kit (Takara). The variable region gene of VHH was amplified by two rounds of PCR, and the gene was inserted into a phage display vector to construct an antibody immunotherapy library.

[0541] Immunological Library Selection

[0542] Biotin-labeled antigen PD-L1-His (ACRO, CAT#PD1-H82E5) was used to enrich specific antibodies targeting PD-L1 through two rounds of panning. Phages selected from each round were then infecting TG1 cells, plated to form monoclonal colonies, and selected for induced expression. The supernatant was then used for ELISA detection. The supernatant was incubated with PD-L1 antigen coated on an ELISA plate at room temperature for 1 hour, followed by incubation with Anti-Flag / HRP secondary antibody (Sigma, A8592) for 45 minutes. TMB was used for color development, and the reaction was terminated with stop solution. The absorbance at OD450 nm was measured; monoclonal colonies with a reading greater than 0.1 were considered positive and used for blocking function screening.

[0543] 3.2 Screening of PD-L1 VHH blocking antibodies

[0544] PD-L1 aAPC cells were seeded in 96-well plates and cultured overnight. Diluted bacterial supernatant and PD1-NFAT-Reporter (Promega, Cat.#J1250, J1255) cells were added to the plates, and the plates were cultured for 6 hours. Chromogenic substrate was added to the plates, and the plates were incubated at room temperature. The luminescence value was measured using a multi-mode microplate reader. Bacterial clones with blocking function were sent to Kingwiz for sequencing to obtain the variable region antibody sequence.

[0545] As shown in Table 5, the supernatant of the candidate PD-L1 VHH antibody in this study showed good PD-L1 blocking activity, with an EC50 of 20.38 nM.

[0546] Table 5: Detection of PD-L1 VHH blocking activity

[0547] Antibody chAb2 EC50 (nM) 20.38

[0548] 3.3 Expression, purification, and screening of VHH candidate PD-L1 antibodies

[0549] The gene sequence of the VHH antibody with PD-L1 blocking function obtained in the above experiments was retrieved and constructed into a chimeric antibody chAb2-Fc. The sequences corresponding to the antibody light and heavy chains are as follows: the Fc region of the antibody heavy chain is the Fc region of human IgG1 (SEQ ID NO:54), and the C-terminus of VHH is coupled to the N-terminus of CH2 of the antibody Fc via Linker_1 (SEQ ID NO:29). The full-length sequence of chAb2-Fc is SEQ ID NO:30. The variable region and CDR sequence of chAb2 are shown in Table 6 below.

[0550] The specific process is as follows:

[0551] Freshly cultured positive clone cell lines, approximately 5E6 cells per line, were used to extract RNA (Macherey-Nagel, Cat#740984.250). cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on the base sequence located in the 5' FR1 region, and downstream primers were designed based on the base sequence located in the antibody constant region or FR4 region to amplify the antibody heavy chain variable region gene fragment. This fragment was ligated into a T-vector (Mighty TA-cloning Kit, Takara), and single clones were selected for sequencing. The sequencing results were analyzed and compared using MEGA7 software.

[0552] The chAb2 heavy chain variable region gene fragment was processed using homologous recombinase from Nanjing Novizan Biosciences Co., Ltd. II, catalog number: C112-01) was ligated into the pcDNA3.1 vector (chAb2-Fc full-length sequence (SEQ ID NO:30), the plasmid containing the IgG1 subtype Fc region (SEQ ID NO:54, CH2CH3) to obtain the expression plasmid of heavy chain antibody.

[0553] Referring to Example 1.4, light and heavy chain plasmids corresponding to SG01 (US20210101982A1) and Atezolizumab (WO2019077132A1) in the table below were prepared to express the corresponding antibodies for subsequent experiments. The heavy chain constant region sequence is IgG1 (SEQ ID NO:21), and the light chain constant region is Kappa (SEQ ID NO:22).

[0554] Table 6: CDR Heavy Chain Comparison Table for Anti-PD-L1 Antibodies

[0555] chAb2 sequence SG01 sequence Atezolizumab sequence VH 25 33 41 CDRH1 26 34 42 CDRH2 27 35 43 CDRH3 28 36 44 VL: / 37 45 CDRL1 / 38 46 CDRL2 / 39 47 CDRL3 / 40 48 Linker_1 29 / /

[0556] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF#A14351-01). Cell density was checked one day before transfection (viability should be greater than 95%), and the cell density was adjusted to 3E6 cells / mL with fresh Expi293F medium and cultured for another day. On the day of transfection, the cell density was adjusted to 3E6 cells / mL.

[0557] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as transfection buffer, add the plasmid of the heavy chain antibody to be transfected at a ratio of 1 mg / L, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 min, then gently pour the mixture into the Expi293F cell suspension while shaking, place the cells in a shaker and culture under the following conditions: 8% CO2, 36.5℃, 120 rpm.

[0558] After culturing for 16–18 hours, 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), 5 g / L glucose solution, and 2.2 mM Valproic acid sodium salt (Merk, Cat#P4543-100G) were added to the cell suspension. The mixture was gently stirred and incubated for another 7 days at 36.5°C and 120 rpm with 8% CO2. The cells were then harvested. The cell suspension was mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0559] Affinity chromatography was used to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing column was washed with 10 column volumes of 1×PBS to remove non-specifically binding proteins. The packing column was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, filtered for sterilization, and used for subsequent functional analysis after the purity test was qualified.

[0560] 3.4 Affinity detection of chimeric anti-PD-L1 antibody chAb2

[0561] In this study, the binding kinetics (KD) of the antibody-antigen in this invention was determined using biomembrane thin-layer interferometry (BLI). The affinity determination by BLI was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

[0562] Half an hour before the experiment, according to the sample quantity, take an appropriate number of AHC sensors (18-5060, Sartorius) and soak them in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). Dilute the anti-PD-L1 antibody and recombinant human PD-L1 protein (Human PD-L1, ACRO, Cat#PD1-H5258) mentioned above to 100 nM respectively.

[0563] SD buffer, antibody solution, and PD-L1 recombinant protein were added to 96-well black polystyrene microplates (Greiner, 655209), respectively. Detection was performed using a Fortebio Octet Red96e. The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: baseline equilibration 120s, antibody loading and solidification 100s, baseline equilibration 120s, antigen binding 100s, and dissociation 120s; rotation speed 1000 rpm; temperature 30℃. After the experiment, the KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in Table 7 below.

[0564] The kinetic parameters from the affinity experiment show that the chimeric PD-L1 antibody chAb2 has a strong affinity for recombinant human PD-L1 protein, and its binding activity is comparable to that of the control antibody SG01 (SG-559-01 in US20210101982A1).

[0565] Table 7: Affinity of anti-PD-L1 antibody chAb2 to PD-L1

[0566]

[0567] Example 4: Detection of the PD-L1 / PD1 blocking activity of alpaca-derived anti-PD-L1 antibody

[0568] To verify whether alpaca-derived anti-PD-L1 antibodies possess PD-L1 / PD1 blocking activity, we tested their ability to effectively activate human PD1-NFAT-Reporter (Promega, Cat.#J1250, J1255) in vitro. The specific procedure is as follows:

[0569] Add 10% fetal bovine serum (SH30406.05, HYCLONE), 1% penicillin-streptomycin bivalent antibiotic (15140-122, GIBCO), 2 mL of 50 mg / mL Hygromycin B (10687010, INVITROGEN), and 2.5 mL of 50 mg / mL Geneticin to F-12K medium (30-2004, ATCC). TM Selective Antibiotic (10131027, Gibco) was mixed thoroughly to prepare F-12K complete medium for the culture of CHO-K1-PD-L1 cells (GM-C01115, Gibco Biotechnology (Shanghai) Co., Ltd.).

[0570] The CHO-K1-PD-L1 cell density was adjusted to 0.4 × 10⁻⁶ cells using F-12K complete medium. 6 50 μl per well was seeded into an F96 MicroWell microplate (167008, NUNC), and the plate was sealed with 200 μl of sterile PBS. The plate was incubated overnight at 37°C.

[0571] The starting concentrations of IgG1, Atezolizumab, and chAb2 antibodies were adjusted to 200 nM (2X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 10 concentrations, with the last concentration being 0 nM.

[0572] Jurkat-PD1-NFAT-luc cells (Promega, Cat.#J1250, J1255) were adjusted to a density of 1.25 × E6 cells / mL using RPMI 1640 complete medium. The plates were removed from the incubator after being incubated overnight, the medium was aspirated, and 40 μl of the prepared antibody was added to each well. The plates were incubated at 37°C for 30 min.

[0573] Remove the plate and add 40 μl of the pre-treated PD1-NFAT-Reporter cells to each well. Incubate at 37°C for 6 hours. Remove the plate from the incubator and allow it to equilibrate to room temperature. Remove the Bio-Glo Luciferase Assay System kit (G7940, PROMEGA) from the -20°C freezer and allow it to equilibrate to room temperature. Add 80 μl of detection solution to each well and incubate in the dark for 3-5 minutes before detecting the cells using a microplate reader.

[0574] The tests (Table 8) show that the chimeric PD-L1 antibody chAb2 has excellent PD-L1 blocking activity and effectively activates T cell signaling. In terms of activity intensity, chAb2 is slightly stronger than the control antibody atezolimab, with IC50 values ​​of 0.188 nM and 0.257 nM, respectively.

[0575] Table 8: Detection of PD-L1 blocking activity of anti-PD-L1 chimeric antibody chAb2

[0576] Antibody Atezolizumab chAb2 IC50(nM) 0.2573 0.1883

[0577] Example 5: Humanization, preparation and activity detection of alpaca-derived anti-PD-L1 antibody

[0578] The chimeric antibodies obtained from hybridomas are humanized using conventional methods, and the specific steps are as follows:

[0579] ① Determine the CDR ring structure;

[0580] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the phylogenetic sequence database;

[0581] ③ Screen for the most closely matched human lineages and the lowest possible amount of reversion mutations to the heavy and light chains;

[0582] ④ Construct the CDR region of the chimeric antibody onto the human backbone region;

[0583] ⑤ Use sequence and structural features to determine the amino acid positions in the backbone region that maintain CDR function;

[0584] ⑥ Perform a reverse mutation (reverting to the input amino acid type) at the identified important sequence positions;

[0585] ⑦ Optimize amino acids at risk sites.

[0586] The final humanized antibody, hzAb2.1, was thus obtained. Its CDR sequence and heavy chain variable region sequence are shown in the sequence listing and Table 9.

[0587] The heavy chain variable region gene fragment was processed by homologous recombinase from Novizan Pharmaceutical Co., Ltd. II, catalog number: C112-01) was ligated into the pcDNA3.1 vector, the plasmid containing the IgG1 subtype Fc region (SEQ ID NO: 54, CH2CH3). The specific preparation process is described in Example 3.3.

[0588] Table 9: Comparison of Heavy Chain Antibodies Against PD-L1 Antibody chA2 Humanized CDR Mutant Antibody

[0589]

[0590] 5.1 Affinity Detection of Humanized Anti-PD-L1 Antibody

[0591] The affinity of the humanized TROP2 antibody of the present invention was determined using the biofilm thin-layer interferometry (BLI) technique mentioned in Example 3.4. The affinity of the humanized molecule is shown in the table below (Table 10).

[0592] The kinetic parameters from the affinity experiment show that the humanized PD-L1 antibody hzAb2.1 has a strong affinity for recombinant human PD-L1 protein, and its binding activity is comparable to that of the parental chimeric antibody chAb2.

[0593] Table 10: Affinity of humanized PD-L1 antibody hzAb2.1 to human PD-L1

[0594] Sample ID KD(M) kon(1 / Ms) kdis(1 / s) chAb2 4.50E-09 4.23E+05 1.91E-03 hzAb2.1 8.66E-09 3.86E+05 3.34E-03

[0595] 5.2 Detection of endocytic activity of humanized anti-PD-L1 antibody

[0596] HCC1954 (CRL-2338, ATCC) cells were adjusted to a density of 5×E4 cells / ml using RPMI 1640 complete medium (10% fetal bovine serum, 1% penicillin and streptomycin). 50 μl of each cell was seeded into F96 MicroWell microplates (167008, NUNC), and the plates were sealed with 200 μl of sterile PBS. The plates were incubated overnight at 37°C.

[0597] The initial concentrations of IgG1, SG01, chAb2, and hzAb2.1 antibodies were adjusted to 400 nM (4X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 10 concentrations, with the last concentration being 0 nM. The initial concentration of DT3C (CSB-EP360556CQR1, Huamei Biotechnology) was also adjusted to 800 nM (4X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 10 concentrations, with the last concentration being 0 nM.

[0598] Take an equal volume of antibody and DT3C (antibody:DT3C = 1:2), mix thoroughly, and incubate at 37°C for 30 min. Add 50 μl of the incubated mixture to each well of the plated cells, and incubate at 37°C for 3 days. Remove the plate from the incubator and allow it to equilibrate to room temperature. Remove the Luminescent Cell Viability Assay kit (DD1101-02, Vazyme) from the -20°C freezer and allow it to equilibrate to room temperature. Add 100 μl of the assay solution from the kit to each well, protect from light, shake at 150 rpm for 2 min, let stand for 10 min, and then detect the luminescence value using a multi-mode microplate reader (SPARK, TECAN). Calculate the cell viability at each concentration using the formula in Example 1.6.

[0599] like Figure 3 As shown, the humanized PD-L1 antibody hzAb2.1 exhibited good endocytic activity, superior to the parental chimeric antibody chAb2. The IC50 values ​​in the figure indicate that the endocytic effect of hzAb2.1 in the HCC1954 cell line was comparable to that of the positive control antibody SG01.

[0600] 5.3 Detection of the blocking activity of humanized anti-PD-L1 antibody against PD-L1 / PD1

[0601] To verify whether the humanized PD-L1 antibody hzAb2.1 still maintains good PD-L1 / PD1 blocking activity, we tested whether these antibodies could effectively activate human PD1-NFAT-Reporter (Promega, Cat.#J1250, J1255) in vitro. The specific procedure is as described in Example 4. The control PD-L1 positive antibody used in the experiment was Atezolizumab (WO2019077132A1), prepared as above.

[0602] As can be seen from the table below (Table 12), the humanized PD-L1 antibody hzAb2.1 still has good PD-L1 blocking activity, and its strength is better than that of the positive control antibody Atezolizumab.

[0603] Table 12: Reporter's detection of PD-L1 antibody's blocking activity against PD-L1 / PD1

[0604] Antibody Atezolizumab hzAb2.1 IC50(nM) 0.2573 0.1883

[0605] Example 6: Construction and preparation of TROP2xPD-L1 bispecific antibody

[0606] In this study, a TROP2xPD-L1 bispecific antibody BsAb targeting TROP2 and PD-L1 was constructed (Table 13). The bispecific antibody molecule is in a "2+2" form ( Figure 4 (), where the sequence targeting TROP2 comes from clone hzAb1.4, and the sequence targeting PD-L1 comes from clone hzAb2.1. For example... Figure 4 As shown, the Fc fragment of the antibody uses IgG1 Fc, which has the LALA(L234AL235A) mutation. TROP2 is located in the Fab region of the antibody, while PD-L1 is located at the C-terminus of CH3 of the antibody heavy chain. The N-terminus of PD-L1 is coupled to the C-terminus of CH3 through a linker (Linker_2).

[0607] Therefore, the heavy chain sequence of the BsAb bispecific antibody is SEQ ID NO:49, the light chain sequence is SEQ ID NO:50, and the Linker_2 sequence is SEQ ID NO:51.

[0608] Table 13: TROP2xPD-L1 dual antibody combination

[0609] TROP2xPD-L1 dual antibody TROP2 PD-L1 BsAb hzAb1.4 hzAb2.1

[0610] 6.1 Preparation of Bispecific Antibodies

[0611] The bispecific antibody application in this invention is similar to the preparation process of monoclonal antibodies. Two plasmids corresponding to the heavy and light chains are co-transfected into expression cells. The culture supernatant is collected and purified through a series of steps to obtain the final target bispecific antibody molecule. For the specific preparation process, please refer to Example 1.4.

[0612] Specifically, the BsAb light and heavy chain coding gene fragments were respectively processed using homologous recombinase from Novizan Pharmaceutical Co., Ltd. II (Catalogue No.: C112-01) was ligated into the pcDNA3.1 vector to obtain expression plasmids for light and heavy chain antibodies. The expression and purification methods are described in Example 1.4.

[0613] 6.2 TROP2xPD-L1 Bispecific Antibody Affinity Detection

[0614] To characterize the affinity of the bispecific antibodies BsAb at both ends, this study used thin-layer interferometry (BLI) to determine the equilibrium dissociation constant (KD) of the bispecific antibodies of this invention binding to human TROP2 and PD-L1 proteins. The Cynomolgus TROP2 antigen was obtained from Kactusbio (TRP-CM121), and the Cynomolgus PD-L1 antigen was obtained from Yiqiao Biotechnology (90251-C08H-100). Specific methods can be found in Example 1.5.

[0615] Tests revealed (Table 14) that the TROP2 and PD-L1 ends of the bispecific antibody maintained very good affinity.

[0616] Table 14: Affinity assay at both ends of the TROP2xPD-L1 bispecific antibody BsAb

[0617]

[0618] Example 7: In vitro activity assay of TROP2xPD-L1 bispecific antibody

[0619] 7.1 TROP2xPD-L1 Dual Antibody In Vitro PD-L1 Blockade and Activation Detection

[0620] To verify whether the 2+2 form of TROP2xPD-L1 bsAb still maintains good PD-L1 blocking activity, this study continued to detect bsAb activity in PD1-NFAT-Reporter cells, and the specific experimental method is as described in Example 4.

[0621] like Figure 5 As shown, the PD-L1 in TROP2xPD-L1 bsAb effectively blocks the interaction between PD-L1 and PD1, activating NFAT signaling in the T reporter. In terms of activity intensity, BsAb achieves PD-L1 blocking activity comparable to the positive antibody Atezolizumab.

[0622] 7.2 TROP2xPD-L1 dual antibody extracellular endocytosis assay

[0623] To further validate the in vitro activity of the TROP2xPD-L1 bispecific antibody, we examined whether it could be effectively internalized into tumor cells expressing both TROP2 and PD-L1. The details are as follows:

[0624] 10% fetal bovine serum (SH30406.05, HYCLONE) and 1% penicillin-streptomycin antibiotics (15140-122, GIBCO) were added to RPMI1640 (22400-071, Gibco) and mixed thoroughly to prepare RPMI1640 complete medium. The cell lines used in this study were EBC1 (JCRB0820, JCRB cell bank), Bxpc3 (CRL-1687, ATCC), HCC1954 (CRL-2338, ATCC), and Panc0813 (CBP60684, Nanjing Kebai Biotechnology Co., Ltd.).

[0625] First, in this study, the expression levels of the above cells were tested by flow cytometry to determine the expression levels of TROP2 and PD-L1 on the cell surface. FACS buffer was prepared by adding 0.5% BSA (V900933-100G, Sigma) and 2 mM EDTA (15575020, Thermo Fisher) to PBS (70011-044, Gibco) and mixing thoroughly.

[0626] EBC1 (JCRB0820, JCRB cell bank), Bxpc3 (CRL-1687, ATCC), HCC1954 (CRL-2338, ATCC), and Panc0813 (CBP60684, Nanjing Kebai Biotechnology Co., Ltd.) cells were adjusted to a density of 5E5 cells / ml using FACS buffer, and 100 μl was seeded into each well of a 96-well V-plate (FPT019, Beyotime). After centrifugation at 300g for 5 min at room temperature, the supernatant was discarded and the cells were ready for use.

[0627] To test the expression levels of PD-L1 and TROP2 in cells, the following experiments were performed: The concentrations of the test antibodies IgG1, SG01, and hRS7 were adjusted to 10 nM using FACS buffer. 100 μl of the prepared test antibody was added to each well of the cells, mixed thoroughly, and incubated at 4°C for 1 h. The cells were washed twice with FACS buffer and set aside. The concentration of PE anti-human Fc (410708, Biolegend) was adjusted to 20 nM using FACS buffer. 100 μl of this prepared PE anti-human Fc was added to each well of the cells, mixed thoroughly, and incubated at 4°C for 0.5 h. The cells were washed twice with FACS buffer, and the MFI was detected by flow cytometry (Symphony, BD). The formula for calculating the Fold Change is: Use GraphPad Prism 10 to create the graph.

[0628] After testing ( Figure 6 We can see that the human esophageal cancer cell line EBC1 and the human pancreatic cancer cell line BxPC3 have high TROP2 expression levels, but low PD-L1 expression levels. On the surfaces of the human pancreatic cancer cell line PANC-0813 and the human breast cancer cell line HCC1954, TROP2 remains at a high level, while PD-L1 shows a significant increase compared to the previous two cell lines, belonging to the intermediate-to-high expression level.

[0629] Then, the cell density was adjusted to 5E4 cells / ml using RPMI 1640 complete medium, and 50 μl was seeded into each well of an F96 MicroWell microplate (167008, NUNC). The plates were sealed with 200 μl of sterile PBS and incubated overnight at 37°C. The starting concentrations of IgG1, hRS7, and bsAb antibodies were adjusted to 400 nM (4X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 8 concentrations, with the last concentration being 0 nM. The starting concentration of fabzap (IT-51, ATSBIO) was adjusted to 800 nM (4X) using RPMI 1640 complete medium, and then serially diluted 4-fold to a total of 10 concentrations, with the last concentration being 0 nM. An equal volume of antibody was mixed with fabzap (antibody: fabzap = 1:2) and incubated at 37°C for 30 min. Add 50 μl of the pre-incubated mixture to each well of the plated cells and incubate at 37°C for 3 days. Remove the plate from the incubator and allow it to equilibrate to room temperature. Remove the Luminescent Cell Viability Assay kit (DD1101-02, Vazyme) from the -20°C freezer and allow it to equilibrate to room temperature. Add 100 μl of detection solution to each well, protect from light, shake at 150 rpm for 2 min, let stand for 10 min, and then detect the luminescence value using a multi-functional microplate reader (SPARK, TECAN).

[0630] The cell viability at various concentrations is calculated using the following formula: Three-parameter fitting was performed using GraphPad Prism 10, with the fitting formula being Y = Bottom + (Top - Bottom) / (1 + (X / IC50)).

[0631] The results are as follows: In TROP2 high PD-L1 low In the two cell lines EBC1 and BxPC3, due to the low expression level of PD-L1, bsAb exhibited endocytic activity comparable to or slightly stronger than hRS7. In the two cell lines PANC0813 and HCC1954, due to the high expression levels of both TROP2 and PD-L1, bsAb exhibited significantly stronger endocytic activity than hRS7. Figure 6 Overall, bsAb has strong endocytic activity, which can mediate the entry of more toxin molecules into tumor cells, thereby producing a good tumor-killing effect.

[0632] Example 8: Preparation of TROP2 x PD-L1 bispecific antibody conjugate

[0633] The antibody-drug conjugates used in this invention include bsAb-NT3, hRS7-NT3, and the control ADC IgG-NT3. All of the linker-payload NT3s are DNA topoisomerase inhibitors, and their molecular structures are disclosed in WO2021173773A1. The preparation process is shown in Example 4 of WO2021173773A1. NT3 has the following structure.

[0634]

[0635] This study also employed random coupling, and the resulting ADC molecular structure is as follows:

[0636]

[0637] Where Ab is the bispecific antibody BsAb, hRS7, or IgG1 prepared in Example 6, q is an integer from 3 to 5, mainly 4, and the obtained BsAb-NT3 has an average DAR of 4 as determined in the following experiments.

[0638] The specific coupling process is as follows:

[0639] 1. Preparation of BsAb-NT3

[0640] (a) The antibody BsAb prepared according to Example 6 was dissolved in PBS buffer (Gibco, 10010-023).

[0641] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, soluble in water), and allow the reaction mixture to react at room temperature for 2 hours.

[0642] (i) The optimal concentration of BsAb is 5 mg / mL.

[0643] (ii) The optimal molar ratio of TCEP / mAb is 2.0.

[0644] (iii) The optimal temperature for the reaction is 25℃.

[0645] (iv) The optimal pH for the reaction is between 6.0 and 8.0.

[0646] (c) Add an excess of linker-toxin (NT3, dissolved in DMSO) and react it with the antibody reduced in step (a). Incubate the reaction mixture at room temperature for 2 hours.

[0647] (i) The optimal molar ratio of NT3 / BsAb is 6.0.

[0648] (ii) The optimal temperature for the reaction is 25°C.

[0649] Obtain the crude ADC product.

[0650] (d) The crude ADC product was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product BsAb-NT3, with an average DAR value of 4.

[0651] (e) The ADC products were analyzed by RP-HPLC, LC-MS and SEC HPLC to determine the average DAR value and SEC purity.

[0652] The DAR value was calculated using RP-HPLC based on the peak area of ​​each peak at UV 280 nm. The calculated average DAR for BsAb-NT3 was 3.89. The analytical conditions are as follows:

[0653]

[0654] (e) Using SEC-HPLC, the purity of the ADC was obtained based on the peak area ratio of monomer, aggregate, and oligomer at UV280nm. The purity of BsAb-NT3 was calculated to be 98.71%.

[0655] The analysis conditions are as follows:

[0656]

[0657]

[0658] 2. Preparation of IgG1-NT3

[0659] (a) Antibody IgG1 was dissolved in PBS buffer.

[0660] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, soluble in water), and allow the reaction mixture to react at room temperature for 2 hours.

[0661] (i) The optimal concentration of IgG1 for Isotype control is 10 mg / mL.

[0662] (ii) The optimal molar ratio of TCEP / mAb is 2.0.

[0663] (iii) The optimal temperature for the reaction is 25℃.

[0664] (iv) The optimal pH for the reaction is between 6.0 and 8.0.

[0665] (c) Add an excess of linker-toxin (NT3, dissolved in DMSO) and react it with the antibody reduced in step (a). Incubate the reaction mixture at room temperature for 2 hours.

[0666] (i) The optimal molar ratio of NT3 / mAb is 6.0.

[0667] (ii) The optimal temperature for the reaction is 25°C.

[0668] Obtain the crude ADC product.

[0669] (d) The crude ADC product obtained was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IgG1-NT3, with an average DAR value of 4.

[0670] (e) The ADC product was analyzed by RP-HPLC, LC-MS and SEC HPLC to determine the average DAR and SEC purity, under the same conditions as BsAb-NT3. The result was IgG1-NT3 DAR 3.6 purity 99.34%.

[0671] 3. Preparation of hRS7-NT3

[0672] (a) Antibody hRS7 was dissolved in PBS buffer.

[0673] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, soluble in water), and allow the reaction mixture to react at room temperature for 2 hours.

[0674] (i) The optimal concentration of hRS7 is 5 mg / mL.

[0675] (ii) The optimal molar ratio of TCEP / mAb is 2.0.

[0676] (iii) The optimal temperature for the reaction is 25℃.

[0677] (iv) The optimal pH for the reaction is between 6.0 and 8.0.

[0678] (c) Add an excess of linker-toxin (NT3, dissolved in DMSO) and react it with the antibody reduced in step (a). Incubate the reaction mixture at room temperature for 2 hours.

[0679] (i) The optimal molar ratio of NT3 / mAb is 6.0.

[0680] (ii) The optimal temperature for the reaction is 25°C.

[0681] Obtain the crude ADC product.

[0682] (d) The crude ADC product obtained was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product hRS7-NT3, with an average DAR value of 4.

[0683] (e) The ADC product was analyzed by RP-HPLC, LC-MS and SEC HPLC to determine the average DAR and SEC purity. The conditions were the same as for BsAb-NT3. The results were: hRS7-NT3 DAR 4.3 purity 98.42%.

[0684] 4. Preparation of SG01-MMAE

[0685] (a) Antibody SG01 was dissolved in PBS buffer.

[0686] (b) Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, soluble in water), and allow the reaction mixture to react at room temperature for 2 hours.

[0687] (i) The optimal concentration of SG01 is 5 mg / mL.

[0688] (ii) The optimal molar ratio of TCEP / mAb is 2.0.

[0689] (iii) The optimal temperature for the reaction is 25℃.

[0690] (iv) The optimal pH for the reaction is between 6.0 and 8.0.

[0691] (c) An excess of the linker-toxin VcMMAE (MCE, HY-15575, dissolved in DMSO) was added and reacted with the antibody reduced in step (a). The reaction mixture was placed at room temperature for 2 hours.

[0692] (i) The optimal molar ratio of NT3 / mAb is 6.0.

[0693] (ii) The optimal temperature for the reaction is 25°C.

[0694] Obtain the crude ADC product.

[0695] (d) The crude ADC product was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product, with an average DAR value of 4.

[0696] (e) The ADC product was analyzed by RP-HPLC, LC-MS and SEC HPLC to determine the average DAR and SEC purity, under the same conditions as BsAb-NT3, and the result was 99.58%.

[0697] Through the above conjugation process, qualified antibody-conjugate samples are obtained for subsequent experimental procedures.

[0698] Example 9: In vitro activity assay of TROP2xPD-L1 bispecific antibody conjugate bsAb-NT3

[0699] 9.1 In vitro cytotoxicity assay of TROP2xPD-L1 bispecific antibody conjugate bsAb-NT3

[0700] To detect the in vitro activity of the TROP2xPD-L1 bispecific antibody ADC bsAb-NT3, this study examined the cytotoxicity of this molecule in several tumor cells expressing TROP2 or PD-L1. The specific procedure is as follows:

[0701] Add 10% fetal bovine serum (SH30406.05, HYCLONE) and 1% penicillin-streptomycin antibiotics (15140-122, GIBCO) to RPMI1640 (22400-071, Gibco), mix well, and prepare RPMI1640 complete medium.

[0702] The cells used in this study were EBC1 (JCRB0820, JCRB cell bank), Bxpc3 (CRL-1687, ATCC), HCC1954 (CRL-2338, ATCC), and Panc0813 (CBP60684, Nanjing Kebai Biotechnology Co., Ltd.).

[0703] Adjust the cell concentrations of the above-mentioned cells to 50,000 cells / mL using complete culture medium. Seed 50 μL (2500 cells / well) per well into 96-well white-background plates (167008, NUNC) and incubate overnight at 37°C. Dilute the ADC molecule with growth medium, starting at 200 nM (2x), with 5-fold serial dilutions for a total of 9 dilutions, the last concentration being 0 nM. Add 50 μL of the diluted antibody-drug conjugate or control to each well, for a total volume of 100 μL. Seal the wells with 200 μL of PBS and incubate at 37°C for 6 days. Remove the cultured 96-well plates and CTL (DD1101-02, Vazyme) and equilibrate to room temperature. Add an equal volume of CTL (100 μL) to each well. Shake on a plate for 5 min to fully lyse the cell clusters, and incubate at room temperature for 10 min to stabilize the luminescence signal. The culture plates were tested using a microplate reader (Spectra MAX i3x, Molecular Divices), and the cell viability was calculated by reading the A450 value.

[0704] The cell proliferation inhibition rate after 6 days of culture was calculated using the following formula.

[0705] Cell proliferation inhibition rate (%) = a / b*100, where a: the average value of the wells with experimental samples added after 6 days of culture, and b: the average value of the wells with control medium added after 6 days of culture (no ADC molecules added, only medium). The IC50 of the drug was calculated by fitting the data with GraphPadPrism.

[0706] Tests revealed that bsAb-NT3 exhibited strong cytotoxic effects, with activity levels superior to the control ADChRS7-NT3. However, the differences between bsAb-NT3 and hRS7-NT3 varied depending on the cell type and the expression levels of TROP2 or PD-L1.

[0707] In TROP2 high PD-L1 low On two cell lines: human esophageal cancer cell line EBC1 and human pancreatic cancer cell line BxPC3, bsAb-NT3 showed activity levels comparable to or slightly stronger than hRS7-NT3, and both were able to effectively inhibit tumor cell growth. Figure 7 ).

[0708] However, in the human pancreatic cancer cell line PANC-0813 and the human breast cancer cell line HCC1954 (TROP2) high PD-L1 med-high The above tests showed that bsAb-NT3 had a better tumor-suppressive effect than the control molecule hRS7-NT3. Figure 7 ).

[0709] 9.2 Detection of the bystander effect in TROP2xPD-L1 dual antibody-coupled objects

[0710] A significant challenge in the treatment of solid tumors with antibody-drug conjugates (ADCs) is the heterogeneous expression of target antigens in tumor tissues or metastases. This means that tumor cells may simultaneously express, underexpress, or not express the target antigen at all, thus affecting the efficacy of ADCs. The "bystander killing effect" offers a potential solution to this problem. The bystander effect refers to the process during ADC synthesis where small molecule compounds can be linked to antibodies via cleavable linkers. After being endocytosed into the cell membrane, the linkers are cleaved, releasing the small molecule and killing the target cell. Following the death of the target cell, the small molecule compound is released into the intercellular space, further killing non-target cells within a certain range. Because tumor cells exhibit significant differences in target expression levels (tumor heterogeneity), the bystander effect is crucial for effectively killing tumor cells and inhibiting tumor growth.

[0711] TROP2xPD-L1 bispecific antibody ADCs can target cells with high expression levels of the tumor antibody TROP2 or PD-L1, killing target cells and releasing the payload drug. Some payload drugs have good hydrophobic activity, allowing the released payload drug to cross the cell membrane of cells surrounding the target cells, thereby achieving a further killing effect on tumor cells.

[0712] To verify the bystander effect of the TROP2xPD-L1 bispecific antibody ADC bsAb-NT3, two cell lines were used in this study: Colo-205 (Nanjing Kebai Biotechnology Co., Ltd.) and HCC1954 (CRL-2338, ATCC). Both cell lines were cultured in RPMI 1640 (22400-071, Gibco) + 10% FBS (SH30406.05, HYCLONE). Colo-205 cells, which showed low TROP2 and PD-L1 expression levels, were subjected to CellTrace... TM Label with Violet (C3455, Thermo) reagent, incubate at 37°C for 20 minutes, then stop the reaction by adding normal culture medium and wash twice with the medium. Add prepared HCC1954 and Colo-205 cells (3:1) to 24-well plates (NEST), and add antibody-drug conjugates (IgG1-NT3, hRS7-NT3, bsAb-NT3), or control IgG1, or NT1 (NT1 has the following structure, disclosed in WO2021173773A1), or an equal volume of culture medium to the corresponding wells at a final concentration of 10 nM. Incubate the prepared samples at 37°C for 5 days. After culture, collect the cells and use LIVE / DEAD. TM Cells were stained using Invitrogen (L34975). The stained cells were then analyzed by flow cytometry.

[0713] After testing ( Figure 8 We observed that both Colo-205 and HCC1954 cell lines exhibited a strong response to the small toxin molecule NT1. Furthermore, Colo-205 cells, initially insensitive to bsAb-NT3, regained a drug response under the HCC1954-mediated "bystander effect." Compared to the control hRS7-NT3, bsAb-NT3 produced a stronger and equally potent bystander effect, further demonstrating that our TROP2xPD-L1 bispecific antibody ADC possesses a stronger hydrophobic loading, enabling it to more effectively penetrate cell membranes and kill cells.

[0714]

[0715] Example 10: In vivo antitumor activity assay of TROP2xPD-L1 bispecific antibody conjugate

[0716] To further validate the antitumor activity of the PSMAxTROP2 bispecific antibody, we examined the antitumor activity of the TROP2xPD-L1 bispecific antibody ADC bsAb-NT3 in a multi-person tumor model in mice.

[0717] Mice: Immunodeficient mice CB17-SCID (Vitalliwa) aged 6-8 weeks were placed in an SPF experimental environment for 3-5 days before the experiment to allow them to better adapt to their current living environment.

[0718] In all experiments, the long and short diameters of the tumor were measured using vernier calipers twice a week, and the tumor volume (mm3) was calculated from the measurement data. The calculation formula is as follows:

[0719] Tumor volume (mm3) = 0.5 * major diameter (mm) * minor diameter (mm) * minor diameter (mm)

[0720] 10.1 Detection of drug efficacy in a human pancreatic cancer BxPC3 tumor model

[0721] The human pancreatic cancer cell line BxPC3, purchased from ATCC, will be acquired at a rate of 3x10. 6 In mice, tumor cells were subcutaneously implanted in the right ventral region. By day 8, the tumor had grown to 150-180 mm in size. 3 Mice were divided into groups to ensure that the average initial drug volume was approximately the same for each group. On the day of grouping, mice were administered the drug via intraperitoneal injection at a dose of 5 mg / kg or 6 mg / kg, with only one dose administered throughout the experiment. The experimental groups were given ADCs (bsAb-NT3 and hRS7-NT3), while the control group was treated with the same dose of hIgG (Equitech-Bio, 211110-0256).

[0722] From the results ( Figure 9 As can be seen, bsAb-NT3 at a dose of 6 mg / kg exhibited a strong tumor-suppressive effect in this model. It showed an equivalent tumor-suppressive effect to hRS7-NT3 at an equimolar dose (5 mg / kg).

[0723] 10.2 Detection of drug efficacy in a human non-small cell lung cancer H322 tumor model

[0724] Human non-small cell lung cancer cell line H322 (Nanjing Kebai Biotechnology CBP60134) was used at 5x10 6 In mice, tumor cells were subcutaneously implanted in the right ventral region. By day 7, the tumor had grown to 150-180 mm in size. 3Mice were divided into groups to ensure that the average initial drug volume was approximately the same for each group. On the day of grouping, mice were administered the drug via intraperitoneal injection at a dose of 2 mg / kg (equivalent dosage), with only one administration throughout the experiment. The experimental groups were given ADCs (bsAb-NT3 and hRS7-NT3), while the control group was treated with equivalent doses of IgG1-NT3 and hIgG (Equitech-Bio, 211110-0256).

[0725] From the results ( Figure 10 As can be seen, in this small cell lung cancer model, a single dose of low-dose (2 mg / kg) of bsAb-NT3 significantly inhibited tumor cell growth. Compared with the control ADC hRS7-NT3, bsAb-NT3 exhibited better antitumor efficacy.

[0726] 10.3 Detection of drug efficacy in a human breast cancer HCC1954 tumor model

[0727] Human breast cancer cell line HCC1954 (CRL-2338, ATCC) was used at 5x10 6 In mice, tumor cells were subcutaneously implanted in the right ventral region. By day 14, the tumor had grown to 150-180 mm in size. 3 Mice were divided into groups to ensure that the average initial drug volume was approximately the same for each group. On the day of grouping, mice were administered the drugs intraperitoneally at doses of 1 mg / kg and 10 mg / kg (equivalent dosages), with only one administration throughout the experiment. The experimental groups were given ADCs: bsAb-NT3, hRS7-NT3, and SG01-MMAE, while the control group was given hIgG (Equitech-Bio, 211110-0256).

[0728] Testing revealed that at a low dose (1.2 mg / kg) ( Figure 11 A), in this study, bsAb-NT3 showed significantly superior tumor-suppressive effects compared to an equivalent molar amount of hRS7-NT3. In the high-dose group (10 mg / kg) ( Figure 11 B), bsAb-NT3 and the control molecule hRS7-NT3 showed equal and equally significant tumor-suppressive effects, both stronger than Seagen BM's SG01-MMAE.

[0729] 10.4 Detection of drug efficacy in a human breast cancer HCC1954-hPD-L1+ tumor model

[0730] To verify the effect of PD-L1 expression level on the antitumor activity of TROP2xPD-L1 bispecific antibody ADC, human PD-L1 protein was transfected into HCC1954 cells via exogenous transfection in this study to test the antitumor activity of bsAb-NT3.

[0731] Cell line establishment:

[0732] The full-length human PD-L1 (UniProt ID: Q04609-1) gene was constructed into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. This vector, along with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), was co-transfected into HEK293T (ATCC, CRL-3216) cells for viral packaging. Culture supernatants were collected after 48 and 72 hours of culture, and lentiviral concentration was performed using a Lenti-X Concentrator (Takara, Cat#631232, Lot#2109261A). HCC1954 cells (DSMZ, catalog number ACC249) were transfected with concentrated virus, and then cells with different PD-L1 expression levels were sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter) to obtain the stable PD-L1 transfected cell line HCC1954-hPDL1. Expression level testing was performed as described in Example 7.2.

[0733] HCC1954-hPDL1 was used at 5x10 6 In mice, tumor cells were subcutaneously implanted in the right ventral region. By day 14, the tumor had grown to 150-180 mm in size. 3 Mice were divided into groups to ensure that the average initial drug volume was approximately the same for each group. On the day of grouping, mice were administered the drug via intraperitoneal injection at a dose of 3 mg / kg, with only one dose administered throughout the experiment. The experimental group received ADCs: bsAb-NT3 and hRS7-NT3, while the control group received an equivalent dose of hIgG (Equitech-Bio, 211110-0256).

[0734] The results showed that the newly established HCC1954-hPDL1 significantly increased the surface level of PD-L1 cells compared to untransfected cells (12A). In this model, an equal molar amount of bsAb-NT3 showed better tumor suppression compared to the control ADC hRS7-NT3 (12B).

[0735] Example 11: In vivo antitumor activity assay of TROP2xPD-L1 bispecific antibody conjugate combined with PD-L1 antibody

[0736] The PD-L1 molecule in bsAb, the molecule studied in this study, possesses PD-L1 blocking activity, thus effectively activating the immune system and mediating an anti-tumor immune response. To simultaneously detect the killing effect of the ADC, the blocking activity of PD-L1, and their potential synergistic effect, we needed to test the molecule in mice with healthy immune systems. Furthermore, in this study, human TROP2 and PD-L1 molecules were transfected onto the surface of the mouse colon cancer cell line CT26 to ensure that the tested molecule bsAb-NT3 could effectively recognize and act on tumor cells. The experimental procedure is as follows:

[0737] Mice: PD1 and PD-L1 humanized mice Bacb / c (Jicui Yaokang) aged 6-8 weeks were placed in an SPF experimental environment for 3-5 days before the experiment to allow them to better adapt to their current living environment.

[0738] In all experiments, the long and short diameters of the tumor were measured using vernier calipers twice a week, and the tumor volume (mm3) was calculated from the measurement data. The calculation formula is as follows:

[0739] Tumor volume (mm3) = 0.5 * major diameter (mm) * minor diameter (mm) * minor diameter (mm)

[0740] Cell line establishment:

[0741] The full-length genes of human PD-L1 (UniProt ID: Q04609-1) and TROP2 (UniProt ID: P09758) were constructed into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. These vectors, along with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), were co-transfected into HEK293T (ATCC, CRL-3216) cells for viral packaging. The culture supernatants were collected after 48 and 72 hours of culture, and the lentivirus was concentrated using a Lenti-X Concentrator (Takara, Cat#631232, Lot#2109261A). CT26 cells (ATCC) were transfected with concentrated virus, and then cells with different TROP2 and PD-L1 expression levels were sorted using a flow cytometer (MoFlo XDP, Beckman Coulter) to obtain the cell line CT26-TROP2-PDL1 that stably expresses TROP2 and PD-L1.

[0742] CT26-TROP2-PDL1 at 5x10 6In mice, tumor cells were subcutaneously implanted in the right ventral region. By day 14, the tumor had grown to 150-180 mm in size. 3 Mice were divided into groups to ensure that the average initial drug volume was approximately the same for each group. After grouping, mice were administered the drug intraperitoneally on the day of grouping at a dose of 5 or 6 mg / kg. Only one dose was administered throughout the experiment. Dosage groups and dosage guidelines are provided. Figure 13 .

[0743] In this study, PD-L1 antibodies Atezolizumab and bsAb showed a certain amount of tumor-suppressive effect, and the effects were comparable. Figure 13 A). Furthermore, bsAb-NT3 exhibited the best tumor-suppressive activity in this model, superior to hRS7-NT3 combined with Atezolizumab. In terms of survival or better, bsAb-NT3 also showed better performance than other groups, consistent with the trend in antitumor activity. Figure 13 B).

[0744] Example 12: Mouse PK Experiment of TROP2xPD-L1 Bispecific Antibody Conjugate

[0745] Balb / c mice (using the drug "Jicui Yaokang") were injected intravenously via the tail vein with bsAb-NT3 (lot: 2023081001). Blood samples were collected at 0.083h, 0.5h, 2h, 6h, 24h, 48h, 96h, 168h, 336h, and 504h post-administration. The serum was transferred to 1.5ml centrifuge tubes, centrifuged at 4000r / min for 10min, and then stored at -80℃ for analysis.

[0746] Method 1 for detecting total antibodies:

[0747] like Figure 14A. Dilute TROP2 protein (ACRO, cat: TR2-H5223, lot: 2573-2316F1-1B9) to 2 μg / mL with coating buffer (carbonate solution), add 100 μL to each well of the microplate, seal, and incubate overnight at 4°C. Discard the coating protein solution, add 300 μL of washing buffer (0.05% Tween 20) to each well and wash 3 times. Add 200 μL of blocking buffer (5% skim milk) to each well and block at room temperature for 2 h. Discard the blocking buffer, add 300 μL of washing buffer to each well and wash 3 times. Adjust the pH of the diluent (1% BSA) to 5.5, add 100 μL of diluted standard (bsAb-NT3), quality control sample (bsAb-NT3), and mouse serum sample to be tested to each well, and incubate at room temperature for 2 h. Discard the sample solution, add 300 μL of washing buffer to each well and wash 3 times. Dilute anti-hFc-HRP (BETHYL, cat: A80-104P, lot: 97) (anti-Fc) 20,000 times with diluent, add 100 μL to each well, and incubate at room temperature for 1 h. Discard the liquid, add 300 μL of washing buffer to each well and wash 6 times. Add 100 μL of TMB substrate (Solarbio, cat: PR1200, lot: 20230418) to each well and develop color in the dark at room temperature for 5–10 minutes. Add 50 μL of ELISA stop solution to each well, vortex at medium speed for 10 seconds, and read the OD values ​​at 450 nm / 620 nm within 30 minutes.

[0748] Method 2 for detecting total antibodies:

[0749] like Figure 14A. Dilute TROP2 protein (ACRO, cat: TR2-H5223, lot: 2573-2316F1-1B9) to 2 μg / mL with coating buffer (carbonate solution), add 100 μL to each well of the microplate, seal, and incubate overnight at 4°C. Discard the coating protein solution, add 300 μL of washing buffer (0.05% Tween 20) to each well and wash 3 times. Add 200 μL of blocking buffer (5% skim milk) to each well and block at room temperature for 2 h. Discard the blocking buffer, add 300 μL of washing buffer to each well and wash 3 times. Adjust the pH of the diluent (1% BSA) to 5.5, add 100 μL of diluted standard (bsAb-NT3), quality control sample (bsAb-NT3), and mouse serum sample to be tested to each well, and incubate at room temperature for 2 h. Discard the sample solution, add 300 μL of washing buffer to each well and wash 3 times. Dilute biotinylated PD-L1 protein (ACRO, cat: PD1-H82E5, lot: BV1055-96HF1-115) to 2 μg / mL using dilution buffer, add 100 μL to each well of the microplate, and incubate at room temperature for 2 h. Discard the liquid, add 300 μL of washing buffer to each well and wash 3 times. Dilute SA-HRP (Biolegend, cat: 405210, lot: B339457) 20000-fold using dilution buffer, add 100 μL to each well, and incubate at room temperature for 1 h. Discard the liquid, add 300 μL of washing buffer to each well and wash 6 times. Add 100 μL of TMB substrate (Solarbio, cat: PR1200, lot: 20230418) to each well and develop color in the dark at room temperature for 5–10 minutes. Add 50 μL of ELISA stop solution (Beijing Solarbio C1058 20190620) to each well, vortex at medium speed for 10 seconds, and read the OD values ​​at 450 nm / 620 nm within 30 minutes using a microplate reader (USA, Thermo Multiskan FCAS-A1-008).

[0750] ADC detection method:

[0751] Anti-payload antibody 64F1H10 (innovent, lot: 20220424) (anti-NT3) was diluted to 2 μg / mL with coating buffer (carbonate solution), and 100 μL was added to each well of the ELISA plate. After sealing, the plate was incubated overnight at 4°C. Figure 14A) Discard the coated protein solution, add 300 μL of washing buffer (0.05% Tween 20) to each well and wash 3 times. Add 200 μL of blocking buffer (5% skim milk powder) to each well and block at room temperature for 2 h. Discard the blocking buffer, add 300 μL of washing buffer to each well and wash 3 times. Adjust the pH of the diluent (1% BSA) to 5.5, add 100 μL of diluted standard (bsAb-NT3), quality control sample (bsAb-NT3), and mouse serum sample to be tested to each well, and incubate at room temperature for 2 h. Discard the sample solution, add 300 μL of washing buffer to each well and wash 3 times. Dilute the biotinylated TROP2 protein (ACRO, cat: TR2-H82E5, lot: BV3055-91PF1-ZC) to 2 μg / mL with the diluent, add 100 μL to each well of the ELISA plate, and incubate at room temperature for 2 h. Discard the liquid, add 300 μL of washing buffer to each well and wash 3 times. Dilute SA-HRP (Biolegend, cat: 405210, lot: B339457) 20000 times with diluent, add 100 μL to each well, and incubate at room temperature for 1 h. Discard the liquid, add 300 μL of washing buffer to each well and wash 6 times. Add 100 μL of TMB substrate (Solarbio, cat: PR1200, lot: 20230418) to each well and develop color in the dark at room temperature for 5–10 minutes. Add 50 μL of ELISA stop solution to each well, vortex at medium speed for 10 seconds, and read the OD values ​​at 450 nm / 620 nm within 30 minutes.

[0752] The OD sample value is the difference between A450 and A620. The concentration value is obtained by fitting a four-parameter Logistic curve using Thermo's Skanit 3.1 analysis software.

[0753] After testing, we found that bsAb exhibited good in vivo stability. Depending on the detection method, the half-life (T-half) of the bispecific antibody ranged from 153 to 180 hours (Table 15), comparable to that of conventional hIgG antibodies. The half-life of bsAb-NT3 was 122 hours (Table 15), indicating that the bispecific antibody ADC in this study had good overall stability in mice. Compared to bsAb, the bispecific antibody ADC bsAb-NT3 also maintained good overall stability in mice. Figure 14 B).

[0754] Table 15: PK detection in mice using TROP2xPD-L1 bispecific antibody conjugate

[0755]

[0756] sequence list

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

Claims

1. An anti-TROP2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1 or 23, and three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:6 or 24.

2. An anti-TROP2 antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein... The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively.

3. The anti-TROP2 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH), wherein the heavy chain variable region a) comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1; or comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:1; or b) comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:23; or comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:23 and / or The antibody or its antigen-binding fragment includes a light chain variable region (VL), wherein the light chain variable region a) comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6; or comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:6; or b) Contains, or consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:24; or contains, or consists of, the amino acid sequence shown in SEQ ID NO:

24.

4. An anti-TROP2 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein a) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; b) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or c) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:6; d) The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:6; e) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24; or f) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

24.

5. The anti-TROP2 antibody or its antigen-binding fragment according to any one of claims 1-4, further comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is or is derived from the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, for example, the heavy chain constant region. (i) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or (ii) Contains an amino acid sequence with at least 85% or 90% of the amino acid sequence of SEQ ID NO:

21. Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity; or (iii) Contains an amino acid sequence with at least 85% or 90% of the amino acid sequence of SEQ ID NO:

56. Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, and possessing the L234A / L235A mutation; and / or The light chain constant region is derived from or originates from the lambda or Kappa light chain constant region, preferably the Kappa light chain constant region, such as the human lambda or Kappa light chain constant region. (i) Contains an amino acid sequence with at least 85% or 90% of the amino acid sequence of SEQ ID NO:

22. Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or consisting of said amino acid sequences; or (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

22.

6. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain... a) comprising, or consisting of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5; or comprising, or consisting of, the amino acid sequence of SEQ ID NO:5; or b) comprising, or consisting of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:58; or comprising, or consisting of, the amino acid sequence of SEQ ID NO:58; and / or The antibody or its antigen-binding fragment comprises a light chain, wherein the light chain a) comprising, or consisting of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 10; or comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 10; or b) Containing, or consisting of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:59; or containing, or consisting of, an amino acid sequence selected from SEQ ID NO:

59.

7. An anti-TROP2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain and a light chain, wherein... a) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10; or b) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:58, and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:59; Preferably, (i) The heavy chain contains the amino acid sequence of SEQ ID NO:5, and the light chain contains the amino acid sequence of SEQ ID NO:10; (ii) The heavy chain consists of the amino acid sequence shown in SEQ ID NO:5, and the light chain consists of the amino acid sequence shown in SEQ ID NO:10; (iii) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:58, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:59; or (iv) The heavy chain consists of the amino acid sequence shown in SEQ ID NO:58, and the light chain consists of the amino acid sequence shown in SEQ ID NO:

59.

8. The anti-TROP2 antibody or its antigen-binding fragment according to any one of claims 1-7, wherein the antibody is a monoclonal antibody; or the antibody is a humanized antibody or a chimeric antibody; or the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody.

9. The anti-TROP2 antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the anti-TROP2 antibody is a bispecific antibody or a multispecific antibody comprising a first binding specificity against TROP2 and a binding specificity against one or more other antigens, optionally, the other antigen being PD-L1.

10. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, The first antigen-binding region specifically binds to TROP2 and includes VH and VL. The VH includes HCDR1, HCDR2, and HCDR3 as defined in claim 1 or 2, and the VL includes LCDR1, LCDR2, and LCDR3 as defined in claim 1 or 2; or The VH and VL are the VH and VL as defined in claim 3 or 4; The second antigen-binding region specifically binds to PD-L1.

11. The bispecific antibody of claim 10, wherein the first antigen-binding region is the Fab region of the anti-TROP2 antibody as defined in any one of claims 1-8. Optionally, the Fab serving as the first antigen-binding region comprises CH1, wherein CH1 is CH1 derived from IgG1, IgG2, IgG3, or IgG4, preferably CH1 derived from IgG1, for example, CH1. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 57; or (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:57; Optionally, the Fab serving as the first antigen-binding region includes a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, for example, the Kappa light chain constant region. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:22; or (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

22.

12. The bispecific antibody of claim 10 or 11, wherein the bispecific antibody comprises an Fc region, for example, the Fc region is or is derived from the Fc region of IgG1, IgG2, IgG3 or IgG4, such as the Fc region of human IgG1, IgG2, IgG3 or IgG4, for example, the Fc region. (i) Containing or consisting of the amino acid sequence of SEQ ID NO:54; or (ii) Contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:54; Optionally, the Fc region contains mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation, for example, in the Fc region. (i) Containing or consisting of the amino acid sequence of SEQ ID NO:55; or (ii) Contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:55 and has an L234A / L235A mutation; Optionally, the Fc region lacks lysine at the C-terminus.

13. The bispecific antibody according to any one of claims 10-12, wherein the second antigen-binding region is a VHH that specifically binds to PD-L1; Optionally, the VHH that specifically binds to PD-L1 includes The three complementary determining regions (CDRs) contained in the VHH shown in either SEQ ID NO:25 or 31; Optionally, the VHH that specifically binds to PD-L1 includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1, VHH CDR2, and VHH CDR3 respectively contain, or are composed of, the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively.

14. The bispecific antibody of claim 13, wherein the VHH that specifically binds to PD-L1 comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 25 or 31; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:25 or 31.

15. The bispecific antibody according to any one of claims 10-14, comprising a heavy chain and a light chain, wherein... The heavy chain from the N-terminus to the C-terminus comprises or consists of the following: the Fab heavy chain of the anti-TROP2 antibody - Fc region - the VHH that specifically binds to PD-L1; The light chain from the N-terminus to the C-terminus comprises or consists of the following: the Fab light chain of the anti-TROP2 antibody; Optionally, the Fc region is connected to the VHH that specifically binds to PD-L1 via a connector; Optionally, the linker is a linker composed of glycine, such as (G)n, where n = any integer between 5 and 15, such as n = 10 or 11, for example, the linker contains or is composed of the amino acid sequence shown in SEQ ID NO:51; Preferably, the Fab heavy chain of the anti-TROP2 antibody comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or are composed of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the VHH specifically binding to PD-L1 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1, VHH CDR2, and VHH CDR3 respectively comprise SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:

9. The amino acid sequence shown in NO:28, or composed of the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively; Preferably, the Fab heavy chain of the anti-TROP2 antibody comprises VH and VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:24; and the VHH that specifically binds to PD-L1 comprises or is composed of a variable region of the heavy chain, wherein the variable region of the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO:

31.

16. The bispecific antibody of claim 15, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; Optionally, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:49; and the light chain comprises the amino acid sequence shown in SEQ ID NO:50; or the heavy chain consists of the amino acid sequence shown in SEQ ID NO:49 and the light chain consists of the amino acid sequence shown in SEQ ID NO:

50.

17. The bispecific antibody of claim 15 or 16, comprising two heavy chains and two light chains, for example, two identical heavy chains and two identical light chains, or consisting of the heavy chains and light chains.

18. A bispecific antibody that specifically binds to TROP2 and PD-L1, comprising a heavy chain and a light chain, optionally, the bispecific antibody comprising two identical heavy chains and two identical light chains, or consisting of said heavy chains and light chains; The heavy chain comprises the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the light chain comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; Optionally, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:49; and the light chain comprises the amino acid sequence shown in SEQ ID NO:50; or the heavy chain consists of the amino acid sequence shown in SEQ ID NO:49 and the light chain consists of the amino acid sequence shown in SEQ ID NO:

50.

19. A VHH antibody that specifically binds to PD-L1, wherein the VHH antibody comprises The three complementary determinant regions (CDRs) contained in the VHH shown in SEQ ID NO:25 or 31; or The VHH antibody comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:26, VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:27, and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:28, or; or VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:26, VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:27, and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:

28.

20. The VHH antibody of claim 19 that specifically binds to PD-L1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 25 or 31; or (ii) Contains or consists of an amino acid sequence selected from or represented by SEQ ID NO:25 or 31.

21. A heavy chain antibody that specifically binds to PD-L1, comprising the VHH antibody that specifically binds to PD-L1 as described in claim 19 or 20; Optionally, the heavy chain antibody comprises a heavy chain, the heavy chain comprising a VHH antibody that specifically binds to PD-L1 as described in claim 19 or 20, which is linked to a constant region or Fc region of the antibody heavy chain, or is composed of the constant region or Fc region of the heavy chain and the VHH antibody; Optionally, the antibody heavy chain constant region is as defined in claim 5, or the Fc region is as defined in claim 12; Optionally, the heavy chain antibody comprises two dimerized heavy chains, wherein the two heavy chains may be the same or different.

22. The VHH antibody that specifically binds to PD-L1 as described in claim 19 or 20, or the heavy chain antibody that specifically binds to PD-L1 as described in claim 21, wherein the antibody is a chimeric antibody or a humanized antibody.

23. A nucleic acid molecule comprising any one chain encoding the anti-TROP2 antibody of any one of claims 1-9 or an antigen-binding fragment thereof, or a bispecific antibody of any one of claims 10-18, or a VHH antibody that specifically binds to PD-L1 of any one of claims 19, 20 and 22, or any one chain of a heavy chain antibody that specifically binds to PD-L1 of claim 20 or 22, or composed of the polynucleotide.

24. An expression vector comprising the nucleic acid molecule of claim 23, preferably, the expression vector being pCDNA, such as pCDNA3.

1.

25. A host cell comprising the nucleic acid molecule of claim 23 or the expression vector of claim 24, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293F cells.

26. A method for preparing the anti-TROP2 antibody of any one of claims 1-9 or its antigen-binding fragment, or the bispecific antibody of any one of claims 10-18, or the VHH antibody specifically binding to PD-L1 of any one of claims 19, 20 and 22, or the heavy chain antibody specifically binding to PD-L1 of claim 20 or 22, the method comprising culturing a host cell of the nucleic acid molecule of claim 23 or the expression vector of claim 24 under conditions suitable for expression of the polypeptide chain of the antibody or its antigen-binding fragment, and optionally recovering the antibody from the host cell (or host cell culture medium).

27. An immunoconjugate comprising the anti-TROP2 antibody of any one of claims 1-9 or its antigen-binding fragment, or the bispecific antibody of any one of claims 10-18, or the VHH antibody that specifically binds to PD-L1 of any one of claims 19, 20 and 22, or the heavy chain antibody that specifically binds to PD-L1 of claim 20 or 22, preferably an antibody-drug conjugate.

28. An antibody-drug conjugate having the formula (I): Ab-(L-D) p (I) Or its pharmaceutically acceptable salts or solvates, in: L is the connector; D represents a drug, preferably an anti-tumor compound; and p is an integer selected from 1 to 16, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Wherein, Ab in formula (I) is the anti-TROP2 antibody or its antigen-binding fragment as described in any one of claims 1-9, or the bispecific antibody as described in any one of claims 10-18, or the VHH antibody that specifically binds to PD-L1 as described in any one of claims 19, 20 and 22, or the heavy chain antibody that specifically binds to PD-L1 as described in claim 20 or 22. Optionally, the antitumor compound is a cytotoxic agent, such as camptothecin, aureatin, or maytansine.

29. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein D has the formula -QL 2 -L 1 -D 1 The structure, Where Q is either -O- or -S-; L 1 Is it non-existent or -(C1-C)? 10 (alkylene)-; L 2 It does not exist, *-(C1-C 10 alkylene)-C(O)N(R 5 )- or *-(C1-C 10 alkylene)-N(R 5 )C(O)-; where * indicates that the end is covalently connected to Q; and R 5 It is an H or C1-C6 alkyl group. D 1 It has the structure shown in equation (D-1): Where R 1 Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and -SR 4 ;R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 Or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2; R 4 Selected from H or C1-C4 alkyl groups; Preferably, R 1 For H, R 2 It is a C1-C6 alkyl or C1-C6 alkoxy, R 3 The preferred halogen is -F.

30. The antibody-drug conjugate of claim 29 or a pharmaceutically acceptable salt or solvate thereof, wherein D 1 It has the structure shown in equation (D-2): Where R 1 R 2 and R 3 As defined in claim 29; Preferably, where D 1 It has the structure shown in formula (D-3) or formula (D-4):

31. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-30, wherein... -L 2 -L 1 - is -(C1-C6 alkylene)-, *-(C1-C6 alkylene)-C(O)N(R) 5 )-(C1-C6 alkylene)- or *-(C1-C6 alkylene)-N(R) 5 )C(O)-(C1-C6 alkylene)-, where * indicates that the terminal is covalently connected to Q; and R 5 It is H or C1-C6 alkyl; preferably, wherein -L 2 -L 1 - is -(C1-C6 alkylene)-.

32. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-31, wherein -QL 2 -L 1 - is -OCH2-CH2-CH2-CH2-.

33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-32, wherein -L- has the following structure: -ZE-NH-CH2- Where Z is connected to Ab, and -CH2- is connected to Q; Z is selected from Where m a1 and m a2 Integers independently selected from 0 to 20; m is selected from integers from 1 to 10. Preferably, Z is Where m is an integer from 1 to 10; The carbonyl group at the right end of Z is covalently connected to E; E is a peptide residue containing 2-10 amino acids, wherein the peptide residue is optionally substituted by one or more groups selected from C. 1-6 Alkyl and polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z.

34. The antibody-drug conjugate according to claim 33, or a pharmaceutically acceptable salt or solvate thereof. Wherein E is a peptide residue composed of 2, 3, or 4 amino acids, wherein the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally substituted with one carbon atom. 1-6 Alkyl substitution; Preferably, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally substituted with one carbon atom. 1-6 Alkyl substitution.

35. The antibody-drug conjugate of claim 34 or a pharmaceutically acceptable salt or solvate thereof, wherein the substituted glutamine or glutamic acid has the structure shown below: Where R 6 It is an H or C1-C6 alkyl group; Preferred Where R 6 It is H or C1-C6 alkyl.

36. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 35, wherein E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu-Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution; preferably, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala- or Where R 6 It is an H or C1-C6 alkyl group, wherein these E groups are covalently linked to Z via the left-hand N-terminus.

37. The antibody-drug conjugate according to claim 36, or a pharmaceutically acceptable salt or solvate thereof. The -ZE-NH-CH2- has the following structure Its left end connects to part Ab.

38. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-37, wherein the antibody-drug conjugate of formula (I) has the following structure: Ab’-(S-L-D) p (I’) Wherein Ab' is defined as Ab as in claim 28, and L, D and p are defined as in any one of claims 28-37.

39. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-38, having an average DAR value of 2-6, for example 3-5, for example about 3.5, 3.6, 3.7, 3.8, 3.9 or 4.

40. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from... Wherein Ab is the anti-TROP2 antibody or its antigen-binding fragment as described in any one of claims 1-9, or the bispecific antibody as described in any one of claims 10-18, or the VHH antibody that specifically binds to PD-L1 as described in any one of claims 19, 20 and 22, or the heavy chain antibody that specifically binds to PD-L1 as described in claim 20 or 22. q is as defined for p in claim 28. Preferably, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate has an average DAR of 2-6, for example 3-5, for example about 3.5, 3.6, 3.7, 3.8, 3.9 or 4.

41. A pharmaceutical composition comprising an anti-TROP2 antibody as claimed in any one of claims 1-9 or an antigen-binding fragment thereof, or a bispecific antibody as claimed in any one of claims 10-18, or a VHH antibody that specifically binds to PD-L1 as claimed in any one of claims 19, 20 and 22, or a heavy chain antibody that specifically binds to PD-L1 as claimed in claim 20 or 22, an immunoconjugate as claimed in claim 27, or an antibody-drug conjugate as claimed in any one of claims 28-40, or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

42. A pharmaceutical combination comprising an anti-TROP2 antibody as claimed in any one of claims 1-9 or an antigen-binding fragment thereof, or a bispecific antibody as claimed in any one of claims 10-18, or a VHH antibody that specifically binds to PD-L1 as claimed in any one of claims 19, 20 and 22, or a heavy chain antibody that specifically binds to PD-L1 as claimed in claim 20 or 22, an immunoconjugate as claimed in claim 27, or an antibody-drug conjugate as claimed in any one of claims 28-40, or a pharmaceutically acceptable salt or solvation thereof, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

43. A method for preventing or treating tumors, such as cancer, in a subject or for activating the immune system of a subject, the method comprising administering to the subject an effective amount of an anti-TROP2 antibody as described in any one of claims 1-9 or an antigen-binding fragment thereof, or a bispecific antibody as described in any one of claims 10-18, or a VHH antibody that specifically binds to PD-L1 as described in any one of claims 19, 20, and 22, or a heavy chain antibody that specifically binds to PD-L1 as described in claim 20 or 22, an immunoconjugate as described in claim 27, or an antibody-drug conjugate as described in any one of claims 28-40 or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as described in claim 41, or a pharmaceutical combination as described in claim 42; Optionally, the tumor is a TROP2-positive tumor, such as a TROP2-positive cancer. Preferably, the tumor cells or tissues of the cancer have elevated levels (e.g., nucleic acid or protein levels) of TROP2, for example, compared to the corresponding cells or tissues of a healthy individual, or to healthy tissues or healthy cells adjacent to the cancerous tissue of the patient. Or / and the tumor is a PD-L1-positive tumor, such as a PD-L1-positive cancer. Preferably, the tumor cells or tissues of the cancer also have elevated levels (e.g., nucleic acid or protein levels) of PD-L1, for example, compared to the corresponding cells or tissues of a healthy individual, or to healthy tissues or healthy cells adjacent to the cancerous tissue of the patient. For example, the tumor or cancer is selected from pancreatic cancer, esophageal cancer, non-small cell lung cancer, breast cancer, or colon cancer. Optionally, the method further includes administering one or more therapies to the patient, such as treatment modalities and / or other therapeutic agents, preferably including radiotherapy or surgery, or other therapeutic agents including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

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