Bispecific antibody, antibody coupling medicine as well as preparation method and application of bispecific antibody and antibody coupling medicine

Abstract

The invention relates to a bispecific antibody capable of simultaneously targeting Trop2 and c-Met, an antibody coupling medicine and a preparation method and application thereof, in particular to a bispecific antibody capable of simultaneously and specifically binding Trop2 and c-Met or an antigen binding fragment thereof, nucleic acid molecules for coding the bispecific antibody or the antigen binding fragment, a carrier, a host cell, the antibody coupling medicine, a medicine composition and medical application. The invention also relates to a composition containing a first antibody specifically binding to Trop2 and a second antibody specifically binding to c-Met, and a medical application thereof. The antibody coupling drug provided by the invention has excellent binding activity to Trop2 and c-Met proteins, shows relatively good targeting and inhibitory activity to Trop2 and / or c-Met positive tumor cells, and is good in safety.

Description

[0001] Cross Reference to Related Applications

[0002] This application is based on CN application No. 202411055600.8, filed on August 2, 2024, and claims priority to it, the contents of all the applications are incorporated herein in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology drugs, in particular to a bispecific antibody targeting Trop2 and c-Met, an antibody conjugate drug and a preparation method and application thereof. BACKGROUND

[0004] Antibody-drug conjugates (ADC) are a new type of targeted chemotherapy drugs coupled by monoclonal antibodies (Antibody) and small molecule cytotoxic drugs (Drug) through a linker (Linker). They have the strong killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs, and are one of the hotspots in the research and development of anti-cancer drugs in recent years.

[0005] Trop2, full name Trophoblast Cell-Surface Antigen 2 (human trophoblast cell surface glycoprotein antigen 2), is a transmembrane protein. Trop2 is a gene closely related to tumors. It mainly promotes tumor cell growth, proliferation and metastasis by regulating calcium ion signaling pathways, cell cycle protein expression and reducing fibronectin adhesion. Trop2 can also interact with β-catenin in the Wnt signaling cascade, thus affecting the transcription of nuclear oncogenes and cell proliferation. Sacituzumab Govitecan (Trodelvy) developed by Immunomedics is the first Trop2 ADC on the market, which is composed of an antibody targeting Trop2 and the active metabolite SN-38 of the chemotherapy drug irinotecan, with a DAR of 7.6. Preclinical data show that even though the DAR value of Trodelvy is as high as 7.6, in Trop2-positive human gastric cancer xenografts (NCI-N87), a dosing dose of 25 mg / kg twice a week for two weeks can only delay tumor growth, and cannot achieve elimination. Therefore, it is necessary to explore more effective, lower dosing dose, and lower dosing frequency of Trop2-targeted drugs.

[0006] c-Met, short for cellular-mesenchymal epithelial transition factor, is a member of the receptor tyrosine kinase family. The c-Met signaling pathway plays a crucial role in invasive growth during embryonic development and postnatal organ regeneration. However, the c-Met signaling pathway can be frequently activated by cancer cells, promoting tumor formation, invasive growth, and metastasis. Studies have shown that the c-Met signaling pathway is abnormally expressed or mutated in various types of solid tumors, such as lung cancer, gastric cancer, liver cancer, breast cancer, skin cancer, and colorectal cancer. Currently, the most advanced c-Met-targeting ADC globally is AbbVie's Telisotuzumab Vedotin, which was approved for marketing in May 2025. It uses a cleavable linker to connect the anti-c-Met monoclonal antibody ABT-700 to the microtubule inhibitor MMAE, with a DAR of 3.1. On January 4, 2022, the FDA granted Telisotuzumab Vedotin Breakthrough Therapy Designation (BTD) for the treatment of patients with advanced / metastatic epidermal growth factor receptor (EGFR) wild-type non-squamous non-small cell lung cancer (NSCLC) who have experienced disease progression during or after platinum-based therapy. Other c-Met ADCs are still in earlier clinical stages, with companies including Hengrui, Rongchang Bio, Regeneron, and Eli Lilly involved in the field.

[0007] Although the efficacy of ADCs has been widely validated, obstacles such as the large size of monoclonal antibodies and low penetration rate in solid tumors still need to be addressed. Nanobodies are small antibodies of only about 15 kDa isolated and screened from the serum of camel species. They contain only the variable domain of heavy chain antibody (VHH), but they have advantages such as high affinity, stability, good water solubility, and strong tissue penetration. The excellent properties of nanobodies have broken through the application bottleneck caused by the large size and complex structure of traditional antibodies, and are expected to bring better efficacy and safety to ADCs.

[0008] Compared to monospecific antibody-drug conjugates (ADCs), bispecific antibody-drug conjugates represent a more novel concept. On one hand, bispecific antibodies can target tumor cells more specifically, overcoming drug resistance and reducing side effects. On the other hand, cross-linking promotes synergistic internalization of the two targets, increasing the efficiency of toxin entry into tumor cells. Furthermore, by reducing the expression of receptor proteins on the cell membrane, they can inhibit tumor cell growth signals, achieving better therapeutic effects. Currently, the development of bispecific antibody-drug conjugates is still in its early stages, with the most advanced being in Phase III clinical trials. JSKN003, a novel antibody-drug conjugate (ADC) targeting two HER2 epitopes developed by KNJ Biopharma, is undergoing a Phase III clinical trial for HER2-low expression, unresectable, and / or metastatic breast cancer. BL-B01D1, a first-in-class EGFR*HER3 bispecific antibody-drug conjugate developed by Baili Tianheng, has initiated a Phase III clinical trial for nasopharyngeal carcinoma, while other indications are mostly in Phase II clinical trials. Currently, there are no publicly reported bispecific antibody-drug conjugates (ADCs) targeting Trop2 and c-Met. Through database analysis, the inventors discovered that Trop2 and c-Met are co-expressed in pancreatic cancer, lung cancer, cervical cancer, breast cancer, urothelial carcinoma, ovarian cancer, and other indications. Furthermore, Trop2 and c-Met exhibit a staggered expression trend in normal tissues. Developing a bispecific antibody-drug conjugate that simultaneously targets Trop2 and c-Met would enhance the tumor targeting of Trop2 and c-Met bispecific antibody-drug conjugates (ADCs) and help solve the toxicity problem of antibody-drug conjugates. Summary of the Invention

[0009] This application discloses a bispecific antibody simultaneously targeting Trop2 and c-Met, wherein the antibody portion can be a modified antibody derived from Trop2 and c-Met nanobodies derived from alpacas. This application also discloses a site-specific antibody-drug conjugate targeting Trop2 and c-Met, consisting of a targeting portion, a cytotoxic drug, and a linker. In some embodiments, this application uses the antimitotic agent monomethylolpropamine E (MMAE, a microtubule inhibitor) as a toxic payload, which is linked to the antibody's site-specific mutation site via a lysosomal cleavable dipeptide valine-citrulline (Val-Cit), thereby obtaining the antibody-drug conjugate. The bispecific antibody-drug conjugate disclosed in this application exhibits good binding activity to Trop2 and c-Met proteins, good targeting at tumor sites in Trop2 and c-Met double-positive or single-positive tumor-bearing mouse models, effectively inhibiting mouse tumor growth, demonstrating highly efficient antitumor activity, and showing good safety.

[0010] Based on the above, the technical solution of this application involves the following aspects.

[0011] 1. Bispecific antibody

[0012] In one aspect, the present invention provides a bispecific antibody that specifically binds to Trop2 and c-Met, comprising a first antigen-binding domain specific to Trop2 and a second antigen-binding domain specific to c-Met.

[0013] Trop2 binding domain

[0014] The first antigen-binding domain specific to Trop2 contained in the bispecific antibody of the present invention can be any antibody form.

[0015] In some embodiments, the first antigen-binding domain is selected from nanobodies, full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab).

[0016] In some embodiments, the first antigen-binding domain is VHH, which includes the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0017] (a) CDR1, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7;

[0018] (b) CDR2, having: the sequence shown in SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:8; and

[0019] (c) CDR3, having: the sequence shown in SEQ ID NO:9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:9;

[0020] In some embodiments, the first antigen-binding domain includes: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9.

[0021] In some embodiments, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1 or a variant thereof; the variant having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0022] In some embodiments, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1.

[0023] c-Met binding domain

[0024] The bispecific antibody of the present invention contains a second antigen-binding domain specific to c-Met, which can be in any antibody form.

[0025] In some embodiments, the second antigen-binding domain is selected from nanobodies, full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab).

[0026] In some embodiments, the second antigen-binding domain is VHH; it comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0027] (a) CDR1, having: the sequence shown in SEQ ID NO:10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:10;

[0028] (b) CDR2, having: the sequence shown in SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:11; and

[0029] (c) CDR3, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12.

[0030] In some embodiments, the second antigen-binding domain includes: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12.

[0031] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:2 or a variant thereof; the variant having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0032] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:2.

[0033] In some embodiments, the second antigen-binding domain is VHH; it comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0034] (a) CDR1, having: the sequence shown in SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:22;

[0035] (b) CDR2, having: the sequence shown in SEQ ID NO:23, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:23; and

[0036] (c) CDR3, having: the sequence shown in SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:24.

[0037] In some embodiments, the second antigen-binding domain includes: CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:23, and CDR3 as shown in SEQ ID NO:24.

[0038] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:19 or a variant thereof; the variant having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived, or having one or more (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) substitutions, deletions, or additions compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0039] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:19.

[0040] The CDR sequences mentioned above can be defined using the Kabat, IMGT, Chothia, or Abm numbering systems.

[0041] Structure of bispecific antibody

[0042] As an example, a bispecific nanobody is provided, wherein both the first antigen-binding domain and the second antigen-binding domain are VHH.

[0043] In some embodiments, the bispecific antibody comprises: (i) a peptide chain IA comprising a first antigen-binding domain, an optional first peptide linker, a second antigen-binding domain, and a monomer of the first Fc domain; and (ii) a peptide chain IB comprising a first antigen-binding domain, an optional second peptide linker, a second antigen-binding domain, and a monomer of the second Fc domain. Exemplary structures are described below. Figure 1 .

[0044] In some embodiments, (i) the peptide chain IA comprises, from N-terminus to C-terminus, the following: a first antigen-binding domain, an optional first peptide linker, a second antigen-binding domain, and a monomer of the first Fc domain; (ii) the peptide chain IB comprises, from N-terminus to C-terminus, the following: a first antigen-binding domain, an optional second peptide linker, a second antigen-binding domain, and a monomer of the second Fc domain; See exemplary structures for details. Figure 1 ① in the middle.

[0045] In some embodiments, (i) the peptide chain IA comprises, from N-terminus to C-terminus, the following components in sequence: a second antigen-binding domain, an optional first peptide linker, the first antigen-binding domain, and a monomer of the first Fc domain; (ii) the peptide chain IB comprises, from N-terminus to C-terminus, the following components in sequence: a second antigen-binding domain, an optional second peptide linker, the first antigen-binding domain, and a monomer of the second Fc domain; exemplary structures are shown below. Figure 1 ② in the middle.

[0046] In some embodiments, (i) the peptide chain IA comprises, from N-terminus to C-terminus, the following: a first antigen-binding domain, a first Fc domain monomer, an optional first peptide linker, and a second antigen-binding domain; (ii) the peptide chain IB comprises, from N-terminus to C-terminus, the following: a first antigen-binding domain, a second Fc domain monomer, an optional second peptide linker, and a second antigen-binding domain; See exemplary structures for details. Figure 1 ③ in the middle.

[0047] In some embodiments, (i) the peptide chain IA comprises, from N-terminus to C-terminus, the second antigen-binding domain, the first Fc domain monomer, an optional first peptide linker, and the first antigen-binding domain; (ii) the peptide chain IB comprises, from N-terminus to C-terminus, the second antigen-binding domain, the second Fc domain monomer, an optional second peptide linker, and the first antigen-binding domain; exemplary structures are shown below. Figure 1 ④ in the middle.

[0048] In the bispecific antibody of the present invention, the peptide linker can be a rigid peptide linker or a flexible peptide linker. In some embodiments, the peptide linker is a peptide linker containing one or more glycines and / or one or more serines. In some embodiments, the peptide linker is (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, or 4.

[0049] In some implementations, the first peptide linker is the same as the second peptide linker.

[0050] In some implementations, the first peptide linker is different from the second peptide linker.

[0051] In some embodiments, the immunoglobulin Fc domain is the Fc domain of IgG (e.g., the Fc domain of IgG1, IgG2, IgG3, or IgG4); in some embodiments, the first and second Fc domain monomers each comprise a hinge region, CH2, and CH3.

[0052] In some implementations, the first and second Fc domain monomers are wild-type IgG Fc domains or are mutated independently, and the mutations can provide a binding site for therapeutic agents (e.g., cytotoxic drugs).

[0053] In some embodiments, the Fc domain monomer comprises a sequence as shown in SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it.

[0054] In some embodiments, the Fc domain monomer may or may not contain mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C.

[0055] In some embodiments, a disulfide bond is contained between the first and second Fc domain monomers.

[0056] In some implementations, no disulfide bond is present between the first and second Fc domain monomers.

[0057] Optionally, the first and second Fc domain monomers each independently contain modifications of one or more amino acids that promote dimerization of the first and second Fc domain monomers. Thus, (hetero)dimerization occurs between the polypeptide containing the first Fc domain monomer and the polypeptide containing the second Fc domain monomer, forming a complex.

[0058] Such modifications are known to those skilled in the art and can include separate modifications to each of the two Fc domain subunits (i.e., the first and second monomers of the Fc domain) to which union is desired, wherein the modifications are complementary to each other, thereby promoting union of the two Fc domain subunits. For example, the union-promoting modifications can alter the structure or charge of one or both Fc domain subunits, thereby promoting their union stereoscopically or electrostatically, respectively. For example, union-promoting modifications include amino acid mutations (e.g., amino acid substitutions) in the Fc domain.

[0059] In some implementations, the modification is in the CH3 domain of the Fc domain.

[0060] In some embodiments, the CH3 domains of the two monomers of the Fc domain contain amino acid substitutions.

[0061] In some embodiments, the modification comprises a “node” modification in one of the two monomers in the Fc domain and a “hole” modification in the other of the two monomers in the Fc domain to form a “knob-into-hole” modification. Knob-into-hole techniques are described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method involves introducing a bulge (“node”) at the interface of a first polypeptide and a corresponding cavity (“hole”) at the interface of a second polypeptide, such that the bulge can be placed within the cavity to promote heterodimer formation and inhibit homodimer formation. The bulge is constructed by replacing the small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity with the same or similar size as the bulge is created in the interface of the second peptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0062] In some exemplary embodiments, an amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue with a larger side chain volume, thereby forming a bulge within the CH3 domain of the first monomer, and an amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue with a smaller side chain volume, thereby forming a complementary cavity with the same or similar size as the bulge within the CH3 domain of the second monomer; or, an amino acid in the CH3 domain of the second monomer is replaced with an amino acid residue with a larger side chain volume, thereby forming a bulge within the CH3 domain of the second monomer, and an amino acid in the CH3 domain of the first monomer is replaced with an amino acid residue with a smaller side chain volume, thereby forming a complementary cavity with the same or similar size as the bulge within the CH3 domain of the first monomer.

[0063] In some embodiments, the amino acid residues with larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (P), tyrosine (Y), and tryptophan (W).

[0064] In some embodiments, the amino acid residues with smaller side chain volumes are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0065] Preferably, the "knob" modification is selected from the following mutations in the EU number: T366W, T366W / S354C, or T366W / S354C / K409A;

[0066] Preferably, the "hole" modification is selected from the following mutations in the EU number: T366S / L368A / Y407V, T366S / L368A / Y407V / Y349C or T366S / L368A / Y407V / Y349C / F405K;

[0067] Preferably, the dimerization-promoting modification includes an L351D / L368E mutation in one of the two Fc domain monomers and an L351K / T366K mutation in the other of the two Fc domain monomers.

[0068] In some embodiments, the bispecific antibody comprises a sequence as shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. In some embodiments, the bispecific antibody has Figure 1 A symmetrical 2+2 structure.

[0069] The sequence shown here does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. Those skilled in the art will understand that during the preparation of proteins through genetic engineering, the first molecule of the resulting polypeptide chain often contains an amino acid (such as Met) encoded by the start codon due to the effect of the start codon. The bispecific antibody of this invention encompasses not only amino acid sequences that do not contain an amino acid (such as Met) encoded by a start codon at their N-terminus, but also amino acid sequences that do contain an amino acid (such as Met) encoded by a start codon at their N-terminus. Therefore, sequences that further contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the aforementioned amino acid sequences are also within the scope of protection of this invention.

[0070] In some embodiments, the bispecific nanobody comprises a variant of the sequence shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29, said variant being a variant of the sequence shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29. NO:29 differs only in one or more (e.g., at most 20, 15, 10, or 5 amino acid substitutions) of conserved amino acid residues, or in having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibody from which it is derived or its antigen-binding fragment, and substantially retains the biological function of the bispecific nanobody from which it is derived (e.g., specific binding to Trop2 and c-Met, neutralizing the biological activity of Trop2 and c-Met). For example, in some embodiments, the variant may be truncated at the N-terminus or C-terminus of the first antigen-binding domain and / or the second antigen-binding domain, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those backbone regions, as long as it substantially maintains antigen binding and specificity.

[0071] In some embodiments, the bispecific antibody is at a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 - 7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with Trop2.

[0072] In some embodiments, the bispecific antibody is at a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 - 7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with c-Met.

[0073] 2. Nucleic acids, vectors, host cells, and methods of expression

[0074] In another aspect, this application provides an isolated nucleic acid molecule comprising a polynucleotide sequence of a bispecific antibody. The nucleic acid can be obtained using methods known in the art, such as isolation from a phage display library, a yeast display library, immunization of animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The nucleic acid molecule can be codon-optimized for the host cell used for expression.

[0075] In another aspect, this application provides a vector containing the said nucleic acid molecule.

[0076] In some embodiments, the nucleic acid molecule is prepared as a recombinant nucleic acid. In some embodiments, the nucleic acid molecule is cloned into an expression vector. The expression vector may further contain additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid containing the nucleic acid can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc. (see Sambrook, J., EFFritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also contain a polynucleotide sequence encoding a polypeptide or protein that facilitates the detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistidine tags (His6), or glutathione S-transferase (GSH) tags), polypeptides containing protease cleavage sites, and reporter proteins (e.g., fluorescent proteins). The nucleic acid molecule may be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacterial, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In still other embodiments, the expression vector is a bacteriophage vector or a phage particle vector.

[0077] In another aspect, this application provides a host cell comprising at least one nucleic acid or vector as described above. In some embodiments, the host cell is used to express the nanobody or its antigen-binding fragment, or a polypeptide construct comprising the nanobody or its antigen-binding fragment. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as *Escherichia coli*), eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Bacteria (e.g., *Escherichia coli* BL21(DE3)) are particularly advantageous for expressing smaller antigen-binding fragments. Suitable mammalian host cells for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0078] 3. Preparation of antibodies

[0079] The antibodies of the present invention can be prepared by various methods known in the art, such as phage surface display technology and genetic engineering recombination technology. For example, DNA molecules encoding the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification, the obtained DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of the present invention. The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules.

[0080] In some implementations, the method includes the following steps:

[0081] (1) Transform host cells using at least one of the nucleic acids or expression vectors described herein;

[0082] (2) Culture the transformed host cells under suitable conditions to allow the expression of the nucleic acid or expression vector, and

[0083] (3) Isolate and purify the antibody or its antigen-binding fragment or the polypeptide construct from the host cell or culture medium.

[0084] In some embodiments, the host cell also contains a chaperone plasmid, which can help improve the solubility, stability, and / or folding of the antibody or antibody fragment. Techniques for isolating and purifying antibodies from host cells are well known to those skilled in the art.

[0085] 4. Conjugates

[0086] On the other hand, this application also provides conjugates comprising the bispecific antibody of the present invention and the conjugated portion.

[0087] In some embodiments, the bispecific antibody of the present invention is optionally conjugated to the conjugation portion via a linker.

[0088] In some embodiments, the conjugation portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose. In some exemplary embodiments, the C-terminus of the bispecific antibody of the present invention is linked to a purification tag.

[0089] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters), magnetic beads (e.g., The labeling includes thermometric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the bispecific antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0090] In some embodiments, the coupling portion is selected from therapeutic agents, such as cytotoxic drugs.

[0091] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0092] 5. Antibody conjugate drugs (ADCs)

[0093] This application further provides an antibody-drug conjugate (ADC) comprising:

[0094] The targeting component is selected from any of the bispecific antibodies described above;

[0095] Cytotoxic drugs section; and

[0096] Connector for connecting the target portion and the cytotoxic drug portion.

[0097] Cytotoxic drugs can be conjugated to disulfide bonds on antibodies, or site-specific and stable conjugation can be achieved using engineered cysteine-mutated conjugate technology. Mutating a specific amino acid site on an antibody to cysteine ​​and reacting it with a drug-linker allows for site-specific conjugation, yielding highly uniform conjugates and improving the therapeutic index of ADC drugs.

[0098] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue.

[0099] In some embodiments, the targeting portion is linked to the linker via a thiol group on a cysteine ​​residue of VHH.

[0100] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue of a reduced disulfide bond in the hinge region.

[0101] In some implementations, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue in the Fc domain.

[0102] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue at position 239 and / or 290 of the Fc domain.

[0103] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors and DNA damage drugs.

[0104] In some embodiments, the microtubule inhibitor is selected from olritamine compounds (e.g., MMAE, MMAF), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4), taxanes (e.g., taxol, docetaxel, carbazitaxel), vinblastines (e.g., vincristine), eribulin, and colchicine.

[0105] In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (cazithromycin γ1l, N-acetyl-γ1I cazithromycin, atrazomycin, PBD, ducamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically camptothecin, SN-38, Dxd, irinotecan, belotetan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone), and muscarinic acid.

[0106] In some embodiments, the cytotoxic drug is MMAE.

[0107] In some implementations, the connector may be a cuttable or non-cuttable connector.

[0108] In some embodiments, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers.

[0109] In some embodiments, the linker is selected from MC (6-maleimide hexanoyl), MCC (maleimide methylcyclohexane-1-carboxylate), MP (maleimide propionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimide)-4-(pyridine-2- (N-succinimide-1-yl)valerate, 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or SIAB (N-succinimide-(4-iodo-acetyl)aminobenzoate) and any combination thereof.

[0110] In some implementations, the connector is MC-Val-Cit-PAB.

[0111] In some embodiments, each peptide chain of the targeting portion is linked to 0, 1, 2, 3, 4, or 5 of the following structures via VHH, cysteine ​​residues in the reduced disulfide bond of the hinge region, or cysteine ​​residues in the Fc domain:

[0112]

[0113] In some implementations, the antibody-drug conjugate is selected from:

[0114]

[0115] Where x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0116] Ab is any of the bispecific antibodies described above.

[0117] In some implementation schemes, the antibody-drug conjugate is:

[0118]

[0119] Where x = 1, 2, 3, 4, 5 or 6;

[0120] Ab contains or is composed of the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

[0121] 6. Compositions

[0122] In another aspect, this application provides a composition comprising or consisting of one or more of the antibody-drug conjugates described in any of the preceding claims.

[0123] In some embodiments, the DAR value of the composition is 1-10, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3, 2- 3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5 2.5-7, 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4.5-5, 4.5-5.5, 4.5- 6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-8.5, 4.5-9, 4.5-9.5, 4.5-10, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5-8.5, 5-9, 5-9.5, 5-10, 5.5-6, 5.5-6.5, 5 .5-7, 5.5-7.5, 5.5-8, 5.5-8.5, 5.5-9, 5.5-9.5, 5.5-10, 6-6.5, 6-7, 6-7.5, 6-8, 6-8.5, 6-9, 6-9.5, 6-10, 6.5-7, 6.5-7.5, 6.5-8, 6.5-8.5, 6.5-9, 6. 5-9.5, 6.5-10, 7-7.5, 7-8, 7-8.5, 7-9, 7-9.5, 7-10, 7.5-8, 7.5-8.5, 7.5-9, 7.5-9.5, 7.5-10, 8-8.5, 8-9, 8-9.5, 8-10, 8.5-9, 8.5-9.5, 8.5-10, 9-9.5, 9-10, 9.5-10...

[0124] In another aspect, this application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0125]

[0126] Where x = 1, 2, 3, 4, 5, 6, 7 or 8;

[0127] Ab contains or is composed of the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

[0128] In some embodiments, the DAR value of the composition is 1-8, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1. 5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4 0.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8 or 7.5-8.

[0129] In some embodiments, the composition has a DAR value of 1-5, for example 3.5-4.5, or even 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0130] 7. Pharmaceutical compositions

[0131] In another aspect, this application provides a pharmaceutical composition comprising any of the bispecific antibodies, nucleic acid molecules, carriers, host cells, conjugates, antibody-drug conjugates, compositions, and optionally carriers or excipients as described in any of the preceding claims.

[0132] The excipient may be one described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavoring agents, sweeteners, and coloring agents.

[0133] In some embodiments, suitable buffers include calcium bicarbonate, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts or combinations thereof.

[0134] In some embodiments, suitable preservatives include antioxidants such as α-tocopherol and ascorbate, and antimicrobial agents such as parabens, chlorobutanol, and phenol. Antioxidants may further include EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine, etc.

[0135] In some implementations, suitable binders include starches such as potato starch, corn starch, and wheat starch; sugars such as sucrose, glucose, dextrose, lactose, and maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, and ethyl cellulose; polyvinylpyrrolidone (polyvinylpyrrolidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, and water, or combinations thereof.

[0136] In some implementations, suitable lubricants include metal stearate salts (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearoyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl betaine, mineral oil, paraffin wax, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal dodecyl sulfate salts (such as sodium dodecyl sulfate, magnesium dodecyl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0137] In some embodiments, the disintegrant may be a non-effervescent disintegrant. Suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginate, sodium glycolate starch, gums such as agar, guar gum, locust bean gum, ark sylvestris gum, pectin, and tragacanth gum. In some embodiments, the disintegrant may be an effervescent disintegrant. Suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0138] In some implementations, suitable flavoring agents may be selected from cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; fennel oil; eucalyptus oil; vanilla; citrus oils such as lemon oil, orange oil, grape and grapefruit oil; and fruit flavorings, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot flavorings.

[0139] In some implementations, suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as sodium salts; dipeptide sweeteners, such as aspartame; dihydrochalcone compounds, glycyrrhizin; stevia (stevioside); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol, etc.

[0140] In some implementations, suitable colorants include food, pharmaceutical and cosmetic pigments (FD&C), pharmaceutical and cosmetic pigments (D&C), and external pharmaceutical and cosmetic pigments (Ext.D&C).

[0141] In some embodiments, suitable chelating agents include ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid. Monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid; 1,3-diaminopropane-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionic acid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid) hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N N,N′,N′-triacetic acid; ethylenediamine-N,N,N′,N′-tetra(methylenephosphonic acid); O,O′-bis(2-aminoethyl)ethylene glycol-N,N,N′,N′-tetraacetic acid; N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid; 1,6-hexanediamine-N,N,N′,N′-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2- Diaminopropane-N,N,N′,N′-tetraacetic acid; hypozinogenyltriacetic acid; hypozinogenyltripropionic acid; trisodium salt of hypozinogenyltris(methylene phosphate); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]tripentadecanehexahydrobromide; or triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid, etc.

[0142] In some implementations, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0143] In some embodiments, suitable surfactants include polysorbate, sodium lauryl sulfate, sodium stearoyl fumarate, polyoxyethylene alkyl ethers, dehydrated sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, glycol esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0144] The pharmaceutical compositions described herein can be formulated into various dosage forms and administered via a variety of different routes, such as oral, rectal, or parenteral administration. The term "parenteral" as used herein can include intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrasheathal, intravenous, intravitreal, epidural, subcutaneous, inhalation, transdermal, transmucosal, sublingual, buccal, and topical (including epidermal, dermal, enema, eye drops, ear drops, intranasal, and vaginal) administration. In some exemplary embodiments, the route of administration may be by injection, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection. Oral formulations may include capsules, tablets, pouches, pills, sugar tablets, lozenges, powders, and granules, etc.

[0145] 8. Pharmaceutical uses

[0146] In another aspect, this application provides the use of the bispecific antibodies, nucleic acid molecules, vectors, host cells, conjugates, antibody-drug conjugates, compositions, or pharmaceutical compositions described herein for the preparation of a medicament for the prevention and / or treatment in subjects of diseases related to Trop2 and / or c-Met.

[0147] In another aspect, this application provides for the prevention and / or treatment of diseases related to Trop2 and / or c-Met, including administering an effective amount of the bispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-drug conjugate, composition, or pharmaceutical composition described herein to a subject in need of such treatment.

[0148] In some implementations, the Trop2-related and / or c-Met-related disease is a tumor, such as a Trop2 and / or c-Met-positive tumor.

[0149] In some embodiments, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer, such as lung adenocarcinoma), oral squamous cell carcinoma, ovarian cancer (e.g., ovarian epithelial carcinoma), cervical cancer, and bladder cancer.

[0150] In some implementations, the subject is a mammal, such as a human;

[0151] In some implementations, the bispecific antibody, nucleic acid molecule, carrier, host cell, conjugate, antibody-drug conjugate, composition, or pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents.

[0152] 9. Detection uses

[0153] On the other hand, this application provides a method for detecting the presence or content of Trop2 and / or c-Met in a sample, which includes using the bispecific antibody or conjugate of the present invention.

[0154] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0155] In some embodiments, the conjugate used in the method comprises the bispecific antibody of the present invention and a detectable label.

[0156] In some embodiments, the bispecific antibody used in the method is labeled with a detectable tag.

[0157] In some embodiments, the bispecific antibody used in the method does not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) carrying a detectable label to detect the bispecific antibody of the present invention.

[0158] In some implementations, the method includes the following steps:

[0159] (1) Contact the sample with the bispecific antibody or conjugate of the present invention;

[0160] (2) Detect the formation of the complex between the bispecific antibody, conjugate and antigen or detect the amount of the complex.

[0161] The formation of the complex indicates the presence of an antigen or cells expressing an antigen;

[0162] The antigen is selected from Trop2 or c-Met.

[0163] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0164] In some embodiments, the method is used to diagnose whether a subject has a disease related to Trop2 and / or c-Met. In such embodiments, the method may further include the step of comparing the amount of Trop2 and / or c-Met in a sample from the subject with a reference value. The reference value may be the level of Trop2 and / or c-Met in a sample from a subject known not to have a disease related to Trop2 and / or c-Met (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of Trop2 and / or c-Met in a sample from the subject is elevated relative to a negative reference value, it indicates that the subject has a disease related to Trop2 and / or c-Met.

[0165] In some embodiments, the Trop2-related diseases are characterized by elevated Trop2 expression and / or excessive Trop2 activity. In some embodiments, the Trop2-related diseases are tumors, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, and bladder cancer.

[0166] In some embodiments, the c-Met-related diseases are characterized by elevated c-Met expression and / or excessive c-Met activity. In some embodiments, the c-Met-related diseases are tumors, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, and bladder cancer.

[0167] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed tumor tissue, biopsy sections, or fine-needle aspiration tissue), histological preparations, etc.

[0168] On the other hand, the use of the bispecific antibody or conjugate of the present invention in the preparation of a detection reagent for detecting the presence or level of Trop2 and / or c-Met in a sample or for diagnosing whether a subject has a disease related to Trop2 and / or c-Met is provided.

[0169] In some embodiments, the conjugate used to prepare the detection reagent comprises the bispecific antibody of the present invention and a detectable label.

[0170] In some implementations, the bispecific antibody used to prepare the detection reagent is labeled with a detectable tag.

[0171] In some embodiments, the bispecific antibody used to prepare the detection reagent does not carry a detectable label. In such embodiments, the detection reagent may further comprise other reagents (such as a second antibody) capable of detecting the bispecific antibody of the present invention.

[0172] Terminology Definition

[0173] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the procedures described herein, such as molecular genetics, nucleic acid chemistry, cell culture, biochemistry, and cell biology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0174] As used herein, the singular forms of “a” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms “including,” “comprising,” “having,” “containing,” or variations thereof are open-ended, not exclusive or exhaustive.

[0175] As used herein, the term "Trop2" stands for Trophoblast Cell-Surface Antigen 2, a transmembrane protein. Trop-2 is encoded by the TACSTD2 gene located on chromosome 1 and consists of 323 amino acids. The Trop-2 protein structure includes a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The sequence of Trop2 is well known to those skilled in the art, and the amino acid sequence can be found, for example, in NCBI Gene ID: NP_002344.2.

[0176] As used herein, the term "c-Met" stands for cellular-mesenchymal male epithelial transition factor, a member of the receptor tyrosine kinase family. c-Met is a proto-oncogene located on chromosome 7q21eq31. Initially translated into a single-stranded precursor protein in vivo, it undergoes post-translational modification to become a three-dimensional structure linked by disulfide bonds. The mature c-Met molecule consists of a 50 kDa extracellular α-chain and a 140 kDa transmembrane β-chain. The amino acid sequence can be found, for example, in NCBIGeneID: NP_000236.2.

[0177] As used herein, the terms “antibody” and “monoclonal antibody” refer to immunoglobulin molecules that are typically composed of two pairs of polypeptide chains, each pair consisting of a light chain (LC) and a heavy chain (HC). Each chain has a variable region, referred to as the heavy chain variable region (VH) and the light chain variable region (VL). Together, the VH and VL are responsible for binding to the antigen recognized by the antibody. There are five major classes (or isotypes) of heavy chains in mammalian immunoglobulins, which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0178] The antibody variable region comprises a framework region (FR) and hypervariable regions (HVR), collectively known as the complementarity-determining region (CDR). The CDR is primarily responsible for binding to the epitopes of the antigen. VH and VL consist of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The amino acid distribution in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0179] As used herein, the terms "nanobody," "single-domain antibody," "VHH antibody," or "camel antibody" refer to antibodies that lack additional antibody domains and possess a single domain (variable region) capable of specifically binding to an antigen or epitope. Nanobodies include, for example, VHH antibodies. H Domain antibodies, V NAR Antibodies, Camelidae VHH antibodies and V L Domain antibody. V NARAntibodies are produced by cartilaginous fish, such as nurse sharks, wobbegon sharks, spiny dogfish, and bamboo sharks. Cameloid VHH antibodies are produced by a variety of species, including camels, llamas, alpacas, dromedary camels, and guanacos, which produce naturally occurring heavy chain antibodies lacking the light chain.

[0180] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). A bispecific antibody comprises two antigen-binding domains with binding specificity to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. In some cases, the different antigen-binding domains are linked by peptide linkers. The term "bispecific nanobody" refers to a bispecific antibody formed by two nanobodies.

[0181] As used herein, the term "Fc region" or "Fc domain" refers to a portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In some embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0182] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of lymphocytes or by cells transfected with the coding sequence of a single antibody. Monoclonal antibodies can be produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0183] As used herein, the term "conservative variant" refers to a protein containing a conserved amino acid substitution that substantially does not affect or reduce the protein's affinity. For example, nanobodies or peptide constructs that specifically bind to c-Met may include up to one, two, five, ten, or fifteen conserved substitutions and specifically bind to c-Met. Conserved amino acid substitutions of functionally similar amino acids are well known to those skilled in the art. The following six groups are considered examples of amino acids that are conserved substitutions for each other:

[0184] 1) Alanine (A), Serine (S), Threonine (T);

[0185] 2) Aspartic acid (D), glutamic acid (E);

[0186] 3) Asparagine (N), glutamine (Q);

[0187] 4) Arginine (R), Lysine (K);

[0188] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0189] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0190] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0191] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0192] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its target antigen. The binding affinity between two molecules can be measured using Kx. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). DThe smaller the value, the tighter the binding between the two molecules, and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) refers to an antibody with a binding affinity of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. K D The value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0193] As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0194] As used herein, the term "isolated" means that a substance (such as a nucleic acid molecule or polypeptide) is isolated from its source or environment in which it exists, i.e., it does not contain any other components.

[0195] As used herein, the term "vector" is a medium used to introduce exogenous nucleic acids into host cells, whereby the exogenous nucleic acid is amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into chromosomes of the genome. In this paper, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

[0196] As used herein, the term "expression vector" refers to a vector capable of expressing DNA operatively linked to a regulatory sequence (such as a promoter, ribosome binding site) that can influence DNA expression. The regulatory sequence may include promoter and terminator sequences and optionally may include origin of replication, selection markers, enhancers, polyadenylation signals, etc. The expression vector may be a plasmid, phage vector, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0197] As used herein, the term "host cell" is a cell used to receive, maintain, replicate, or amplify a vector. Host cells can also be used to express nucleic acids or polypeptides encoded by the vector. Host cells can be eukaryotic or prokaryotic cells.

[0198] As used in this article, the term "contact" refers to direct physical association; it includes both solid and liquid forms.

[0199] As used herein, the term "cytotoxic drug" refers to any drug or compound capable of killing cells. "Cytotoxicity" refers to the toxicity of a molecule to its intended target cell, not to the cells of the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of a molecule to cells other than its intended target cell.

[0200] As used herein, the terms “subject,” “patient,” or “individual” include both mammals and non-mammals. Mammals can be any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees, apes, or other monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs (or canines), and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and so on. Non-mammals can include birds, fish, etc. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be male. In some cases, the subject can be female. In some cases, the subject has been diagnosed with or is suspected of having a disease. In some cases, the disease is cancer. The subject can be a patient or an individual. In some cases, subject, patient, or individual may be used interchangeably.

[0201] As used herein, the terms “treatment,” “management,” “improvement,” or “relief” include alleviating or reducing the symptoms of a disease, suppressing the disease (e.g., preventing its progression), alleviating the disease, causing the disease to regress, relieving symptoms caused by the disease, or stopping the symptoms of the disease. The terms “treatment,” “management,” “improvement,” or “relief” may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the treated disease. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the treated disease, resulting in an observable improvement in the subject, although in some implementations the subject may still suffer from the underlying disease.

[0202] As used herein, the terms "effective dose" and "therapeutic effective dose" refer to an adequate amount of medication administered that will at least partially relieve the symptoms of the disease being treated. Dosing regimens can be adjusted to provide the optimal desired response. For example, a single bolus injection may be administered, or several fractions may be given over time, or the dose may be reduced or increased proportionally depending on treatment progress. It should be noted that dose values ​​can vary depending on the type and severity of the disease to be alleviated, and may include single or multiple doses. To further understand, for any given individual, the specific dosing regimen should be adjusted over time based on individual needs and the drug's instructions or the professional judgment of a clinician. Generally, the effective dose is approximately 0.0001 to approximately 50 mg per kg of body weight per day, for example, approximately 0.01 to approximately 10 mg / kg / day (single or fractionated doses). For a 70 kg person, this would total approximately 0.007 mg / day to approximately 3500 mg / day, for example, approximately 0.7 mg / day to approximately 700 mg / day. In some cases, a dose level not exceeding the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0203] Advantages of the invention

[0204] This application provides bispecific antibodies that simultaneously target Trop2 and c-Met, as well as antibody-drug conjugates, compositions containing them, and their uses. The antibody-drug conjugates exhibit good binding activity to both Trop2 and c-Met proteins, demonstrate good targeting and inhibitory activity against Trop2 and c-Met double-positive or single-positive tumors, and have good safety profiles. Attached Figure Description

[0205] Figure 1 The structures of different bispecific antibodies targeting Trop2 / c-Met were shown.

[0206] Figure 2 An example of the structure of an ADC (Anti-Trop2 / c-Met B5-VC-MMAE) is shown.

[0207] Figure 3 The band sizes of Anti-Trop2 / c-Met B5 BsAb identified by SDS-PAGE are shown.

[0208] Figure 4 The results show the results of ELISA assays for the binding activity of Anti-Trop2 Ab, Anti-c-Met Ab, Anti-Trop2 / c-Met B5BsAb, and negative control 125s to antigen Trop2.

[0209] Figure 5 The results show the results of ELISA assays for the binding activity of Anti-Trop2 Ab, Anti-c-Met Ab, Anti-Trop2 / c-Met B5BsAb, and negative control 125s to antigen c-Met.

[0210] Figure 6 The results of detecting the DAR value of Anti-Trop2 / c-Met B5-VC-MMAE using hydrophobic interaction chromatography (HIC) are shown.

[0211] Figure 7 The Biocare method was used to determine the affinity of Anti-Trop2 / c-Met B5 BsAb before coupling.

[0212] Figure 8 The results of flow cytometry analysis of the binding and internalization abilities of Anti-Trop2 / c-Met B5 BsAb and parental antibodies in the pancreatic cancer cell line BxPC3 are presented.

[0213] Figure 9 The results of flow cytometry analysis of the binding and internalization abilities of Anti-Trop2 / c-Met B5 BsAb and parental antibodies in the colorectal cancer cell line HT-29 are presented.

[0214] Figure 10 The results of targeting detection of Anti-Trop2 / c-Met B5 BsAb and parental antibodies in BxPC3 tumor-bearing mice with Trop2-high / c-Met-high pancreatic cancer cell line are shown.

[0215] Figure 11 The results of targeting detection of Anti-Trop2 / c-Met B5 BsAb and parental antibody in SPC-A1 tumor-bearing mice with low Trop2 expression / high c-Met expression are shown.

[0216] Figure 12 This study demonstrated the tumor-suppressive effect of a single gradient administration of Anti-Trop2 / c-Met B5-VC-MMAE on the BxPC3 tumor-bearing mouse line, which is highly expressive of Trop2 / c-Met pancreatic cancer cells.

[0217] Figure 13A and Figure 13BThe tumor-suppressing effects and weight changes of single doses of Anti-Trop2 / c-Met B5-VC-MMAE, Anti-Trop2-VC-MMAE, and Anti-c-Met B5-VC-MMAE in NCI-H358, SPC-A1, MDA-MB-231, and HT-29 tumor-bearing mice are shown.

[0218] Figure 14 The results show the detection of the binding activity of humanized bispecific antibodies targeting Trop2 / c-Met with antigen Trop2.

[0219] Figure 15 The results show the detection results of the binding activity of humanized bispecific antibodies targeting Trop2 / c-Met with different structures to the antigen c-Met.

[0220] Figure 16 The study demonstrated the intracellularization levels of humanized bispecific antibodies and parental antibodies targeting Trop2 / c-Met with different structures in the pancreatic cancer cell line BxPC3, as detected by flow cytometry.

[0221] Figure 17 The study demonstrated the intracellularization levels of humanized bispecific antibodies and parental antibodies targeting Trop2 / c-Met with different structures in the human lung adenocarcinoma cell line SPC-A-1, as detected by flow cytometry.

[0222] Figure 18 The internalization of humanized bispecific antibodies targeting Trop2 / c-Met with different structures in the pancreatic cancer cell line BxPC3 was shown by immunofluorescence.

[0223] Figure 19 The results show the detection results of the binding activity of Anti-Trop2 / c-Met A4 BsAb with antigens Trop2 and c-Met using ELISA.

[0224] Figure 20 The cell binding and internalization levels of Anti-Trop2 / c-Met A4 BsAb in pancreatic cancer cell lines BxPC3, panc02.03, SW1990, and HPAC were shown by flow cytometry.

[0225] Figure 21A and Figure 21B The study showed the tumor-suppressing effect and weight changes in BxPC3 tumor-bearing mice after gradient administration of Anti-Trop2 / c-Met A4-VC-MMAE.

[0226] Sequence information

[0227] Information about the sequences involved in this invention is described in the table below:

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234] Detailed Implementation

[0235] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0236] Example 1: Site-directed modification and design of Trop2 and c-Met parental peptide constructs

[0237] 1.1 In the early stages of the laboratory, nanobodies targeting Trop2 and c-Met were screened from the alpaca antibody library based on phage display and screening techniques. The amino acid sequences are shown in Table 1. The underlined sequences are the CDR sequences (SEQ ID NO:7~SEQ ID NO:12).

[0238] Table 1. Amino acid sequence of parental VHH

[0239]

[0240]

[0241] 1.2 The screened Anti-Trop2 and Anti-c-Met nanobodies were tandemly expressed with the Fc segments CH2 and CH3 (SEQ ID NO:3) of a conventional antibody to extend their half-life. The expression vector used was PTT5-H, with serine at position 239 mutated to cysteine ​​and lysine at position 290 mutated to cysteine ​​(underlined), and named "Anti-Trop2 Ab" and "Anti-c-Met Ab B5" respectively. See Table 2.

[0242] Table 2. Amino acid sequences of Anti-Trop2 Ab and Anti-c-Met Ab B5 (with Fc tags)

[0243]

[0244]

[0245] 1.3 Anti-Trop2 VHH and Anti-c-Met VHH, along with the Fc segments CH2 and CH3 (SEQ ID NO:3) of a traditional antibody, were expressed in tandem using the PTT5-H expression vector. Serine at position 239 was mutated to cysteine, and lysine at position 290 was mutated to cysteine ​​to obtain the Anti-Trop2 / c-Met polypeptide construct, named "Anti-Trop2 / c-Met B5 BsAb", as shown in Table 3.

[0246] Table 3. Amino acid sequences of Anti-Trop2 / c-Met B5 BsAb (with Fc tag)

[0247]

[0248] Example 2: Transient expression and affinity chromatography purification of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5 BsAb

[0249] 2.1 Plasmid Acquisition: Anti-Trop2 Ab, Anti-c-MetAb B5, and Anti-Trop2 / c-Met B5 BsAb plasmids were synthesized by General Biosystems (Anhui) Co., Ltd. The plasmids were rapidly transferred into DH-5α Escherichia coli competent cells (Shenzhen Kangti), heat-shocked for 90 seconds, and then cultured overnight at 37°C in ampicillin-resistant LB agar. Single colonies were picked and amplified in ampicillin-resistant LB agar at 37°C, 220 rpm for 15 hours. Plasmids were then extracted using an endotoxin-free plasmid extraction kit.

[0250] 2.2 Antibody Expression: Expi-293F cells were transiently transfected with single plasmids to express Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5 BsAb. The prepared density was 4 × 10⁻⁶ cells / cells. 6200 mL of Expi-293F cells with a viability of 95% were cultured. 0.5 mg of each antibody plasmid was filtered through a 0.22 μm filter and added to 5 mL of CD05 medium. Simultaneously, 2 mg of PEI was added to 5 mL of CD05 medium, vortexed immediately for 8 seconds, and incubated for 2 minutes. Then, 7 mL of the PEI mixture was added to the plasmid mixture, vortexed immediately for 8 seconds, and incubated for 8 minutes. Finally, the mixture was pipetted dropwise into 200 mL of cell culture medium, gently mixing as it was added. The cells were cultured in a 5% CO2, 37°C shaker for 4 hours. After 4 hours, 200 mL of freestyle medium was added, and the cells were cultured again in a shaker for 7-8 days for expression.

[0251] 2.3 Antibody Purification: Collect the cell expression supernatant of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5BsAb, and centrifuge at 10,000 rpm for 25 min. Filter the cell supernatant through a 0.22 μm filter for later use.

[0252] AKTA purification instrument operation: Set the software flow rate to 8 mL / min and the maximum pressure to 0.3 MPa. First, thoroughly flush the instrument tubing with solution B (100 mM citric acid monohydrate), reduce the flow rate to 2 mL / min, load the protein A medium-pressure chromatography column, and equilibrate the protein A medium with 95% solution A (200 mM disodium hydrogen phosphate dodecahydrate) at a flow rate of 8 mL / min until the baseline level stabilizes, which takes about 15 min. Load the sample at a flow rate of 8 mL / min. The UV value will rise and remain at a certain level; this peak is the breakthrough peak. After loading the sample, equilibrate again with 95% solution A. The peak value will drop to the baseline level and stabilize. Elute with 70% solution B. During this process, the peak value will first rise and then drop to the baseline. The formation of the elution peak is the elution of the target protein. Collect the eluent from this process. Rinse the tubing for contaminating proteins with 100% solution B, then fill the tubing and protein A column with 20% ethanol. Remove the column and store it at 4°C. The eluted proteins were dialyzed into 20 mM PBS via a dialysis bag at 4°C for 24 h. Antibody concentration was measured using a microplate reader or BCA. If the antibody concentration was <0.5 mg / mL, it was concentrated using a 10 kDa Millipore concentrator at 3000 rpm for 10 min. The antibodies were aliquoted and stored at -20°C for later use.

[0253] Figure 3The image shows the band sizes of Anti-Trop2 / c-Met B5 BsAb identified by SDS-PAGE. As shown in the figure, under non-reducing conditions, the band sizes of the three antibodies, Anti-Trop2 / c-Met B5 BsAb, Anti-Trop2 Ab, and c-Met Ab B5, are approximately 120 kDa, 90 kDa, and 80 kDa, respectively. Under reducing conditions, the band sizes of the three antibodies are approximately 60 kDa, 45 kDa, and 40 kDa, respectively, which are close to the theoretical values.

[0254] Example 3: Preparation of Anti-Trop2 / c-Met B5-VC-MMAE

[0255] Using the antimitotic agent monomethylolpropamine E (MMAE, a microtubule inhibitor) as a toxic payload, it is linked to the aforementioned site-specific mutation site of the antibody via a lysosomal cleavable MC-Val-Cit-PAB (maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl) linker, thereby obtaining an antibody-drug conjugate (such as...). Figure 2 (As shown). The detailed experimental steps are as follows:

[0256] (1) Add 0.5M EDTA to the antibody reaction system to make its working concentration reach 5mM;

[0257] (2) Reduction: Add 10 eq of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; BEYOTIME) to the reaction system and incubate at 37°C for 2 h. The disulfide bonds on the engineered antibody and the cysteine-activated thiol groups on the mutation sites are exposed. Replace the buffer through an ultrafiltration concentrator (10Kd; Millipore).

[0258] (3) Oxidation: Add excess Dehydroascorbic acid (DHAA) (50 eq), incubate at room temperature for 3 h, and re-oxidize and link the reduced disulfide bonds, exposing only the engineered cysteine ​​sites. Use an ultrafiltration concentration tube to replace the buffer and remove the oxidant DHAA.

[0259] (4) Conjugation: MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) was dissolved in DMSO at a storage concentration of 10 mM. The added DMSO accounted for 10% of the total reaction system. The mixture was vortexed and incubated at 4°C. 6 eq of MC-vc-PAB-MMAE was added to the reaction system of the Anti-Trop2 / c-Met B5BsAb engineered antibody and incubated at 4°C for 4 h. Excess small molecules, cysteine, and impurities such as DMSO were removed using an ultrafiltration concentrator. The antibody-drug conjugate Anti-Trop2 / c-Met B5-VC-MMAE was obtained.

[0260] The parental antibody-drug conjugates Anti-Trop2-VC-MMAE and Anti-c-Met B5-VC-MMAE were obtained using the same reaction system.

[0261] Example 4: ELISA detection of the binding activity of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5BsAb with antigen Trop2.

[0262] Trop2 antigen (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated into 96-well plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. Anti-Trop2 Ab, Anti-c-Met Ab B5, Anti-Trop2 / c-Met B5 BsAb, and the negative control 125s were diluted to different concentrations and added to 96-well plates pre-coated with Trop2 antigen (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37°C for 30 min, washed 5 times with PBST, and 100 μg / well of substrate chromogenic solution A / B was added. After approximately 10 min of development, a blue gradient was observed. Then, 50 μL / well of stop solution was added, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that Anti-Trop2Ab and Anti-Trop2 / c-Met B5 BsAb bound to antigen Trop2 in a concentration-dependent manner, with EC50... 50 All were at the ng / mL level (as shown in Table 4), Anti-c-Met Ab B5, and the negative control showed no specific binding trend at 125s (e.g. Figure 4 (As shown).

[0263] Table 4. EC50 values ​​(Trop2) of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5 BsAb.

[0264]

[0265] Example 5: ELISA detection of the binding activity of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5BsAb to antigen c-Met.

[0266] Antigen c-Met (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated into 96-well plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. Anti-Trop2 Ab, Anti-c-Met Ab B5, Anti-Trop2 / c-Met B5 BsAb, and the negative control 125s were diluted to different concentrations and added to 96-well plates pre-coated with c-Met antigen (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37°C for 30 min, washed 5 times with PBST, and 100 μg / well of substrate chromogenic solution A / B was added. After approximately 10 min of development, a blue gradient was observed. Then, 50 μL / well of stop solution was added, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that Anti-c-MetAb B5 and Anti-Trop2 / c-Met B5 BsAb bound to antigen c-Met in a concentration-dependent manner, with EC50... 50 All were at the μg / mL level (as shown in Table 5). Anti-Trop2 Ab and the negative control showed no specific binding trend at 125s (e.g., ...). Figure 5 (As shown).

[0267] Table 5. EC50 values ​​(c-Met) of Anti-Trop2 Ab, Anti-c-Met Ab B5, and Anti-Trop2 / c-Met B5 BsAb.

[0268]

[0269] Example 6: Identification of the mean DAR value of Anti-Trop2 / c-Met B5-VC-MMAE by hydrophobic interaction chromatography (HIC)

[0270] (1) A silica-based HPLC column (4.6×100mm, 3.5μm, Agilent) was used for HIC-UPLC (waters) analysis to determine the drug-antibody ratio (DAR);

[0271] (2) Antibodies and their conjugates were eluted via a linear gradient for 40 min from buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), pH 7.5, 0.5 mL / min, 25 °C. Figure 6 As shown: Anti-Trop2 / c-Met B5-VC-MMAE has a DAR of 4 at pH 7.5, which is consistent with the theoretical value.

[0272] Example 7: Affinity determination of Anti-Trop2 / c-Met B5 BsAb before and after coupling using the Biocare method

[0273] A CM5 chip is used to conjugate Trop2 antigen or c-Met antigen. Different concentrations of antibodies or ADCs are injected into the system and flow through the chip to bind with the antigen. The chip surface is dissociated using 3M MgCl2. (Biacore) TM The 8K (GE Healthcare Life Sciences) system calculates the affinity constant. The affinity curve is shown below. Figure 7 As shown in Table 6, the affinity values ​​are all in the nM range, and the conjugation of small molecule drugs did not affect the affinity of the antibody.

[0274] Table 6. KD(M) values ​​of Anti-Trop2 / c-Met B5 BsAb and ADC

[0275]

[0276] Example 8: Flow cytometry detection of the internalization capacity of Anti-Trop2 / c-Met B5 BsAb in BxPC3 and HT-29 cells

[0277] The pancreatic cancer cell line BxPC3, which highly expresses Trop2 and c-Met, and the colorectal cancer cell line HT-29, which lowly expresses Trop2 and highly expresses c-Met, were selected. BxPC3 and HT-29 (2×10⁻⁶ cells / year) were then used as the primary in vitro cytokine (PLC) lines. 5The antibody (10 μg / mL) was resuspended in pre-chilled PBS, and three replicates were prepared. The samples were incubated at 4°C for 1 hour, centrifuged at 1200 rpm for 3 minutes, and washed. Two replicates were taken and resuspended in 2% FBS 1640, incubated at 37°C for 30 minutes and 3 hours respectively, centrifuged at 1200 rpm for 3 minutes, and washed. The secondary antibody (Goat anti-Human IgG FcCross-Adsorbed Secondary Antibody, DyLight) was then added. TM 650 (Invitrogen) 1:400 dilution, incubated at 4°C for 30 min, and analyzed by loading onto an analytical flow cytometer (LSRFortessaX-20; BD).

[0278] like Figure 8 , Figure 9 As shown, the binding rate of Anti-Trop2 / c-Met B5 BsAb in the BxPC3 cell line was significantly higher than that of the parental antibody, and the binding rate in the HT-29 cell line was also superior to that of the parental antibody. After 3 hours of incubation, the internalized fluorescence intensity of Anti-Trop2 / c-Met B5 BsAb in the BxPC3 cell line was more than 200% of that of the parental antibody, and the internalized fluorescence intensity in the HT-29 cell line was more than 160% of that of the parental antibody.

[0279] Example 9: In vivo targeting of Anti-Trop2 / c-Met BsAb in tumor-bearing mice

[0280] 9.1 In vivo targeting of Anti-Trop2 / c-Met B5 BsAb in Trop2-high / c-Met-high pancreatic cancer BxPC3 tumor-bearing mice

[0281] The antibody was coupled with Cyanine 5.5 NHS ester (Luminex Life Science Solutions) at a ratio of 1:10, mixed by 4° rotation overnight, and then desalted using a Zeba column. TM Spin Desalting Columns,7K MWCO,0.5mL; Thermo Scientific TM Centrifugation was used to remove free luciferin. Nude mice (5-6 weeks old, female) were selected, and BxPC3 was administered at a rate of 2 × 10⁻⁶. 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Mice were grouped uniformly according to tumor size, and each mouse was injected with 200 μg of antibody via tail vein. Small animal fluorescence in vivo imaging (Caliper IVI Slimina II) was performed on days 1, 3, 5, and 7. Figure 10As shown: Anti-Trop2 / c-Met B5 BsAb showed significant enrichment at the tumor site in tumor-bearing mice on day 1, with less enrichment in other organs. The parental antibody, due to its weaker targeting than the bispecific antibody, showed significant distribution in the liver and kidneys for the first three days, and concentrated enrichment at the tumor site on day 5. On day 7, the mice were dissected, and the heart, liver, spleen, lungs, kidneys, and tumors were removed. Fluorescence signals in each organ and tumor site were detected. It was observed that both Anti-Trop2 Ab and Anti-Trop2 / c-Met B5 BsAb were concentrated enriched at the tumor site, but the enrichment fluorescence intensity of Anti-Trop2 / c-Met B5 BsAb was significantly stronger than that of Anti-Trop2 Ab, while Anti-c-Met Ab B5 was enriched in both the liver and tumor sites.

[0282] 9.2 In vivo targeting of Anti-Trop2 / c-Met B5 BsAb in SPC-A1 lung adenocarcinoma-bearing mice with low Trop2 expression / high c-Met expression

[0283] The antibody was coupled with Cyanine 5.5 NHS ester (Luminex Life Science Solutions) at a ratio of 1:10, mixed by 4° rotation overnight, and then desalted using a Zeba column. TM Spin Desalting Columns,7K MWCO,0.5mL; Thermo Scientific TM Centrifugation was used to remove free luciferin. Nude mice (5-6 weeks old, female) were selected, and SPC-A1 was applied at a rate of 2 × 10⁻⁶. 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Mice were grouped uniformly according to tumor size, and each mouse was injected with 200 μg of antibody via tail vein. Small animal fluorescence in vivo imaging (Caliper IVI Slimina II) was performed on days 1, 3, 5, and 7. Figure 11 As shown: Anti-Trop2 / c-Met BsAb B5 and parental antibodies were clearly distributed in the liver for the first three days, and concentrated in the tumor site on the fifth day; on the 7th day, the mice were dissected, and the heart, liver, spleen, lung, kidney and tumor were removed. The fluorescence signals of each organ and tumor site were detected. Since SPC-A1 is a cell line with low Trop2 expression and high c-Met expression, Anti-cMet Ab B5 and Anti-Trop2 / c-Met BsAb B5 were concentrated in the tumor site, while Anti-Trop2 Ab was concentrated in the tumor tissue and liver tissue.

[0284] Example 10: Single-needle tumor-suppressing effect of Anti-Trop2 / c-Met B5-VC-MMAE

[0285] 10.1 Single-injection gradient tumor inhibition effect of Anti-Trop2 / c-Met-VC-MMAE in BxPC3 pancreatic cancer tumor-bearing mice with high Trop2 / c-Met expression

[0286] Select nude mice (5-6 weeks old, female), and BxPC3 at a ratio of 2×10 6 Cells were subcutaneously seeded until the tumor volume reached 200 mm². 3 Tumors were uniformly grouped according to size, with the following groups: Anti-Trop2 / c-Met B5-VC-MMAE 10 nmol / kg, 12.5 nmol / kg, 15 nmol / kg, 20 nmol / kg, and PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0287] The tumor size was measured using calipers, and its volume was calculated using the following formula: V = (W / W) 2 ×L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 2000 mm 3 The mice were euthanized. Figure 12 As shown: At a dose of 20 nmol / kg, Anti-Trop2 / c-Met B5-VC-MMAE can almost completely remove 200 mmHg. 3 The growth of BxPC3 tumors was observed, while no decrease in body weight or other abnormalities were observed in any of the groups of animals.

[0288] 10.2 Single-injection antitumor effect of Anti-Trop2 / c-Met B5-VC-MMAE on NCI-H358, SPC-A1, MDA-MB-231 and HT-29 tumor-bearing mice

[0289] Nude mice (5-6 weeks old, female) were selected, and NCI-H358, SPC-A1, MDA-MB-231, and HT-29 were used in a regimen of 2 × 10⁻⁶. 6 Cells were subcutaneously inoculated until the tumor volume reached 100–200 mm. 3 Tumors were uniformly grouped according to size, with the following groups: Anti-Trop2 / c-Met B5-VC-MMAE 20 nmol / kg, Anti-Trop2-VC-MMAE 20 nmol / kg, Anti-c-Met-VC-MMAE 20 nmol / kg, and PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0290] The tumor size was measured using calipers, and its volume was calculated using the following formula: V = (W / W) 2 ×L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 1500 mm... 3 The mice were euthanized. Figure 13A As shown: At a dose of 20 nmol / kg, Anti-Trop2 / c-Met B5-VC-MMAE can completely remove 100–200 mmHg. 3 Anti-Trop2-VC-MMAE and Anti-c-Met-VC-MMAE only inhibited tumor growth, showing a significant difference compared to Anti-Trop2 / c-Met B5-VC-MMAE. Meanwhile, no decrease in body weight or other abnormalities were observed in any of the groups of animals. Figure 13B ).

[0291] Example 11: Construction, expression, and purification of humanized bispecific antibodies Anti-Trop2 / c-Met B5BsAb with different structures

[0292] Following the methods in Examples 1 and 2, humanized bispecific antibodies with different structures were prepared, such as... Figure 1 As shown, they are respectively:

[0293] ①Anti Trop2 / c-Met B5 BsAb, from N-terminus to C-terminus, contains Anti-Trop2 VHH, Anti-c-Met B5 VHH, and the traditional antibody Fc segment CH2 and CH3;

[0294] ②Anti c-Met B5 / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-c-Met B5VHH, Anti-Trop2 VHH, and the traditional antibody Fc segment CH2 and CH3;

[0295] ③Anti-Trop2 / c-Met B5 BsAb, from N-terminus to C-terminus, contains Anti-Trop2 VHH, traditional antibody Fc segment CH2, CH3, and Anti-c-Met B5 VHH;

[0296] ④Anti c-Met B5 / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-c-Met B5VHH, traditional antibody Fc segment CH2, CH3, and Anti-Trop2 VHH.

[0297] The amino acid sequences of humanized Anti-Trop2 VHH and humanized Anti-c-Met B5VHH are shown in SEQ ID NO:13 and SEQ ID NO:14.

[0298] Table 7. ①Anti Trop2 / c-Met B5 BsAb, ②Anti c-Met B5 / Trop2 BsAb,

[0299] ③Amino acid sequences of Anti Trop2 / c-Met B5 BsAb and ④Anti c-Met B5 / Trop2 BsAb

[0300] (with Fc tag)

[0301]

[0302]

[0303]

[0304] Example 12: Binding activity of humanized bispecific antibodies Anti-Trop2 / c-Met B5BsAb with different structures to antigen Trop2

[0305] Referring to the method in Example 4, the binding activity of humanized bispecific antibodies targeting Trop2 / c-Met with different structures to the Trop2 antigen was detected. Figure 14 As shown, humanized bispecific antibodies with different structures all have certain binding activity with antigen Trop2. Among them, antibody ① and antibody ② have higher binding activity with antigen Trop2.

[0306] Table 8. EC50 values ​​(Trop2) of ①Anti Trop2 / c-Met B5 BsAb, ②Anti c-Met B5 / Trop2 BsAb, ③AntiTrop2 / c-Met B5 BsAb, and ④Anti c-Met B5 / Trop2 BsAb.

[0307]

[0308] Example 13: Binding activity of humanized bispecific antibodies Anti-Trop2 / c-Met B5BsAb with different structures to antigen c-Met

[0309] Referring to the method in Example 5, the binding activity of humanized bispecific antibodies targeting Trop2 / c-Met with different structures to the antigen c-Met was detected. Figure 15As shown, humanized bispecific antibodies with different structures all have certain binding activity with antigen c-Met. Among them, antibody ① and antibody ② have higher binding activity with antigen c-Met.

[0310] Table 9. EC50 values ​​(c-Met) of ①Anti Trop2 / c-Met B5 BsAb, ②Anti c-Met B5 / Trop2 BsAb, ③AntiTrop2 / c-Met B5 BsAb, and ④Anti c-Met B5 / Trop2 BsAb.

[0311]

[0312] Example 14: Flow cytometry detection of the internalization ability of humanized bispecific antibodies in BxPC3 and SPC-A1 cells

[0313] The pancreatic cancer cell line BxPC3, which highly expresses Trop2 and c-Met, and the lung adenocarcinoma cell line SPC-A1, which lowly expresses Trop2 and highly expresses c-Met, were selected. BxPC3 and SPC-A1 (2×10⁻⁶ cells / year) were then used as the primary in vitro cytokine cell lines. 5 The antibody (10 μg / mL) was resuspended in pre-chilled PBS in two replicates. The samples were incubated at 4°C for 1 hour, centrifuged at 1200 rpm for 3 minutes, and washed. One replicate was resuspended in 2% FBS 1640, incubated at 37°C for 3 hours, centrifuged at 1200 rpm for 3 minutes, and washed. The secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight) was then added. TM 650 (Invitrogen) 1:400 dilution, incubated at 4°C for 30 min, and analyzed by loading onto an analytical flow cytometer (LSRFortessaX-20; BD).

[0314] like Figure 16 , Figure 17 As shown, four different humanized bispecific antibody ADCs with different structures all exhibited significant binding ability and internalization effects in BxPC3 and SPC-A1 cell lines. In comparison, the "2+0" structural features of ①Anti-Trop2 / c-Met B5 BsAb and ②Anti-c-Met B5 / Trop2 BsAb showed better efficacy and were more suitable for constructing bispecific ADCs.

[0315] Example 15: Immunofluorescence characterization of the cell internalization ability of bispecific antibodies

[0316] Figure 18The study demonstrated the internalization of humanized bispecific antibodies targeting Trop2 / c-Met with different structures in the pancreatic cancer cell line BxPC3 using immunofluorescence.

[0317] On day 1, pancreatic cancer cell line BxPC3 was seeded at 10,000 cells / well and cultured overnight. On day 2, primary antibodies were incubated at two intervals: 3 hours and 0.5 hours. Specifically, on day 1, pancreatic cancer cell line BxPC3 was seeded at 10,000 cells / well and cultured overnight. On day 2, primary antibodies were incubated at two intervals: ① Anti-Trop2 / c-Met B5 BsAb, ② Anti-c-Met B5 / Trop2 BsAb, ③ Anti-Trop2 / c-Met B5 BsAb, and ④ Anti-c-Met B5 / Trop2 BsAb. The dilution buffer was 2% FBSDMEM, and the final antibody concentration was 100 nM / L. Fixation: Wash three times with PBS, add 100 μL of 4% formaldehyde solution, and incubate for 20 min. Permeabilization: Wash three times with PBS, add 100 μL of immunofluorescence permeabilization buffer, and incubate for 5 min. Blocking: Wash twice with PBS, add 100 μL of 2% BSA, and incubate for 30 min. Add secondary antibody: Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody), diluted 1:2000, and incubate at 4°C for 30 min. Stain cell nuclei: Wash three times with PBS, add 100 μL of 1:2000 diluted DAPI (inverted to mix), and incubate at room temperature for 10 min. Image using a high-content cell screening imaging system (Opera Phenix). Figure 18 As shown: at 0.5h, ①Anti-Trop2 / c-Met B5 BsAb showed a significant internalization effect in the BxPC3 cell line, while the other three bispecific antibodies did not show significant internalization; at 3h, all four bispecific antibodies with different structures showed significant internalization.

[0318] Example 16: Site-directed modification and design of Trop2 and c-Met parental peptide constructs

[0319] 16.1 In the early stages of the laboratory, nanobodies targeting Trop2 and c-Met were screened from the alpaca antibody library based on phage display and screening techniques. The amino acid sequences are shown in Table 10. The underlined sequences are the CDR sequences (SEQ ID NO:7~SEQ ID NO:9, SEQ ID NO:22~SEQ ID NO:24).

[0320] Table 10. Amino acid sequence of parental VHH

[0321]

[0322] 16.2 The screened Anti-Trop2 and Anti-c-Met nanobodies were tandemly expressed with the Fc segments CH2 and CH3 (SEQ ID NO:3) of a conventional antibody to extend their half-life. The expression vector used was PTT5-H, with serine at position 239 mutated to cysteine ​​and lysine at position 290 mutated to cysteine ​​(underlined), named "Anti-Trop2 Ab" and "Anti-c-Met Ab A4" respectively. See Table 11.

[0323] Table 11. Amino acid sequences of Anti-Trop2 Ab and Anti-c-Met Ab A4 (with Fc tags)

[0324]

[0325]

[0326] 16.3 Anti-Trop2 VHH and Anti-c-Met A4 VHH, along with the Fc fragments CH2 and CH3 (SEQ ID NO: 3) of the traditional antibody, were expressed in tandem using the PTT5-H expression vector. Serine at position 239 was mutated to cysteine, and lysine at position 290 was mutated to cysteine ​​to obtain the Anti-Trop2 / c-Met A4 polypeptide construct, named "Anti-Trop2 / c-MetA4 BsAb", as shown in Table 12.

[0327] Table 12 Amino acid sequences of Anti-Trop2 / c-Met A4 BsAb (with Fc tag)

[0328]

[0329]

[0330] Example 17: Construction of humanized bispecific antibodies with different structures

[0331] Following the methods in Examples 1 and 2, humanized bispecific antibodies with different structures were prepared, namely:

[0332] ①Anti-Trop2 / c-Met A4 BsAb, from N-terminus to C-terminus, contains Anti-Trop2 VHH, Anti-c-Met A4 VHH, and the traditional antibody Fc segment CH2 and CH3;

[0333] ②Anti c-Met A4 / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-c-Met A4VHH, Anti-Trop2 VHH, and the traditional antibody Fc segment CH2 and CH3;

[0334] ③Anti Trop2 / c-Met A4 BsAb, from N-terminus to C-terminus, contains Anti-Trop2 VHH, traditional antibody Fc segment CH2, CH3, and Anti-c-Met A4 VHH;

[0335] ④Anti c-Met A4 / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-c-Met A4VHH, traditional antibody Fc segment CH2, CH3, and Anti-Trop2 VHH.

[0336] The amino acid sequence of the humanized Anti-c-MetA4 VHH is shown in SEQ ID NO:25.

[0337] Table 13. Amino acid sequences of ①Anti-Trop2 / c-Met A4 BsAb, ②Anti c-Met A4 / Trop2 BsAb, ③AntiTrop2 / c-Met A4 BsAb, and ④Anti c-Met A4 / Trop2 BsAb.

[0338] (with Fc tag)

[0339]

[0340]

[0341]

[0342] Example 18: ELISA detection of the binding activity of Anti-Trop2 / c-Met A4 BsAb with antigens Trop2 and c-Met.

[0343] Antigens Trop2 and c-Met (purchased from Sino Biological) were diluted to 200 nM / L with PBS and plated into 96-well plates (100 μL per well). After overnight incubation at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. Anti-Trop2 / c-Met A4BsAb was diluted to a series of different concentrations and added to each of the pre-coated 96-well plates (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37°C for 30 min, washed 5 times with PBST, and 100 μL of a 1:1 mixture of substrate chromogenic solution A / B was added to each well. After approximately 10 min of incubation, a blue gradient was observed. Then, 50 μL of stop solution was added to each well, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that Anti-Trop2 / c-Met A4 BsAb binds to the antigen in a concentration-dependent manner, with EC50... 50 All are in the nM / L range (as shown in Table 14 and Figure 19 (As shown).

[0344] Table 14. EC50 values ​​of Anti-Trop2 / c-Met A4 BsAb

[0345] Trop2 protein c-Met protein EC 50 (nM / L) 4.176 16.61

[0346] Example 19: Flow cytometry detection of the internalization ability of Anti-Trop2 / c-Met A4 BsAb in pancreatic cancer BxPC3, panc02.03, SW1990 and HPAC cells.

[0347] Pancreatic cancer cell lines BxPC3, panc02.03, SW1990, and HPAC were selected. Cells (2 × 10⁻⁶) were... 5 The antibody (10 μg / mL) was resuspended in pre-chilled PBS in two replicates. The samples were incubated at 4°C for 1 hour, centrifuged at 1200 rpm for 3 minutes, and washed. One replicate was resuspended in 2% FBS 1640, incubated at 37°C for 3 hours, centrifuged at 1200 rpm for 3 minutes, and washed. The secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight) was then added. TM 650 (Invitrogen) 1:400 dilution, incubated at 4°C for 30 min, and analyzed by loading onto an analytical flow cytometer (LSRFortessaX-20; BD).

[0348] like Figure 20As shown, the internalization rate of Anti-Trop2 / c-Met A4 BsAb in cell lines ranges from 30% to 70%.

[0349] Example 20: Single-injection gradient tumor inhibition effect of Anti-Trop2 / c-Met A4-VC-MMAE in BxPC3 pancreatic cancer tumor-bearing mice with high Trop2 / c-Met expression.

[0350] Select nude mice (5-6 weeks old, female), and BxPC3 at a ratio of 2×10 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Tumors were uniformly grouped according to size, with the following groups: Anti-Trop2 / c-Met A4-VC-MMAE 5 nmol / kg, 10 nmol / kg, 20 nmol / kg, 25 nmol / kg, and PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0351] The tumor size was measured using calipers, and its volume was calculated using the following formula: V = (W / W) 2 ×L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 1500 mm... 3 The mice were euthanized. Figure 21A As shown: At a dose of 20 nmol / kg, Anti-Trop2 / c-Met B5-VC-MMAE can completely remove 100 mmHg. 3 The growth of BxPC3 tumors was observed, while no decrease in body weight or other abnormalities were observed in any of the groups of animals. Figure 21B ).

[0352] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A bispecific antibody that specifically binds to Trop2 and c-Met, comprising a first antigen-binding domain specific to Trop2 and a second antigen-binding domain specific to c-Met.

2. The bispecific antibody of claim 1, wherein the first antigen-binding domain is VHH, comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7; (b) CDR2, having: the sequence shown in SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:8; and (c) CDR3, having: the sequence shown in SEQ ID NO:9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:9; Preferably, the first antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; Preferably, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

3. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding domain is VHH; comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:10; (b) CDR2, having: the sequence shown in SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:11; and (c) CDR3, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12; Preferably, the second antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12; Preferably, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:2 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

4. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding domain is VHH; comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:22; (b) CDR2, having: the sequence shown in SEQ ID NO:23, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:23; and (c) CDR3, having: the sequence shown in SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:24; Preferably, the second antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:23, and CDR3 as shown in SEQ ID NO:24; Preferably, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:19 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

5. The bispecific antibody according to any one of claims 1-4, further comprising an immunoglobulin Fc domain.

6. The bispecific antibody according to any one of claims 1-5, wherein the bispecific antibody comprises: (i) The peptide chain IA comprises, from the N-terminus to the C-terminus, the following in sequence: the first antigen-binding domain, an optional first peptide linker, the second antigen-binding domain, and the first Fc domain monomer; (ii) The peptide chain IB comprises, from the N-terminus to the C-terminus, the following components in sequence: the first antigen-binding domain, an optional second peptide linker, the second antigen-binding domain, and the second Fc domain monomer; or (i) The peptide chain IA comprises, from the N-terminus to the C-terminus, the following in sequence: the second antigen-binding domain, an optional first peptide linker, the first antigen-binding domain and the first Fc domain monomer; (ii) The peptide chain IB comprises, from the N-terminus to the C-terminus, the following components in sequence: the second antigen-binding domain, an optional second peptide linker, the first antigen-binding domain, and the second Fc domain monomer; or (i) The peptide chain IA comprises, from the N-terminus to the C-terminus, the following in sequence: the first antigen-binding domain, the first Fc domain monomer, an optional first peptide linker, and the second antigen-binding domain; (ii) The peptide chain IB comprises, from the N-terminus to the C-terminus, the following components in sequence: the first antigen-binding domain, the second Fc domain monomer, an optional second peptide linker, and the second antigen-binding domain; or (i) The peptide chain IA comprises, from the N-terminus to the C-terminus, the second antigen-binding domain, the first Fc domain monomer, an optional first peptide linker, and the first antigen-binding domain. (ii) The peptide chain IB comprises, from the N-terminus to the C-terminus, the second antigen-binding domain, the second Fc domain monomer, an optional second peptide linker, and the first antigen-binding domain.

7. The bispecific antibody according to claim 6, wherein the peptide linker is a rigid peptide linker or a flexible peptide linker; Preferably, the peptide linker is a peptide linker containing one or more glycines and / or one or more serines; Preferably, the peptide linker is (G4S)n, where n is an integer not less than 0, such as 1, 2, 3 or 4; Optionally, the first peptide linker may be the same as or different from the second peptide linker.

8. The bispecific antibody according to any one of claims 5-7, wherein the immunoglobulin Fc domain is the Fc domain of IgG (e.g., the Fc domain of IgG1, IgG2, IgG3 or IgG4); preferably, each of the first and second Fc domain monomers comprises a hinge region, CH2 and CH3; Optionally, the first and second Fc domain monomers are wild-type IgG Fc domains or are mutated independently, and the mutations can provide a binding site for therapeutic agents (e.g., cytotoxic drugs). Preferably, the Fc domain monomer comprises a sequence as shown in SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; Preferably, the Fc domain monomer contains or does not contain mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C; Optionally, the first and second Fc domain monomers each independently contain one or more amino acid modifications that can promote the dimerization of the first and second Fc domain monomers; Preferably, the first and second Fc domain monomers may or may not contain disulfide bonds.

9. The bispecific antibody according to any one of claims 1-8, wherein, The bispecific antibody comprises a sequence as shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29; Preferably, the bispecific antibody has the structure described in any one of claims 6-8.

10. The bispecific antibody according to any one of claims 1-9, wherein the bispecific antibody is at a concentration of less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with Trop2; and / or The bispecific antibody is at a concentration of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with c-Met.

11. An isolated nucleic acid molecule encoding the bispecific antibody as described in any one of claims 1-10.

12. A vector comprising the nucleic acid molecule of claim 11; preferably, the vector is a cloning vector or an expression vector.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing the bispecific antibody according to any one of claims 1-10, comprising culturing the host cell according to claim 13 under conditions that allow protein expression, and recovering the bispecific antibody from the cultured host cell culture.

15. A conjugate comprising the bispecific antibody as described in any one of claims 1-10 and the conjugated moiety; Preferably, the coupling portion is selected from protein tags, such as purification tags; detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin; therapeutic agents, such as cytotoxic drugs; or other bioactive peptides.

16. Antibody-drug conjugates (ADCs), comprising: The targeting portion is selected from the bispecific antibodies described in any one of claims 1-10; Cytotoxic drugs section; and Connector for connecting the target portion and the cytotoxic drug portion.

17. The antibody-drug conjugate of claim 16, wherein the bispecific antibody is linked to the linker via a thiol group on a cysteine ​​residue; Preferably, the bispecific antibody is linked to the linker via a thiol group on a cysteine ​​residue in the VHH or Fc domain, or a thiol group exposed by a cysteine ​​residue in a reduced disulfide bond in the hinge region. Preferably, the bispecific antibody is linked to the linker via a thiol group on a cysteine ​​residue in the VHH, the cysteine ​​residue in the reduced disulfide bond of the hinge region, or the cysteine ​​residue at position 239 and / or 290 of the Fc domain.

18. The antibody-drug conjugate of claim 16 or 17, wherein the cytotoxic agent is selected from microtubule inhibitors and DNA damage agents; Preferably, the microtubule inhibitor is selected from olistatin compounds (e.g., MMAE, MMAF), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4), taxanes (e.g., taxol, docetaxel, carbazitaxel), vinblastines (e.g., vincristine, vinblastine), eribulin, and colchicine; Preferably, the DNA damaging agent is selected from DNA alkylating agents (cazithromycin γ1l, N-acetyl-γ1I cazithromycin, atrazomycin, PBD, ducamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically camptothecin, SN-38, Dxd, irinotecan, belotetan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone), and muscarine; Preferably, the cytotoxic drug is MMAE.

19. The antibody-drug conjugate according to any one of claims 16-18, wherein the linker is a cleavable or non-cleavable linker; Preferably, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers; Preferably, the linker is selected from MC (6-maleimide hexanoyl), MCC (maleimide methylcyclohexane-1-carboxylate), MP (maleimide propionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimide)-4-(pyridin-2-ylthio) 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or SIAB (N-succinimide-(4-iodo-acetyl)aminobenzoate) and any combination thereof; Preferably, the connector is MC-Val-Cit-PAB.

20. The antibody-drug conjugate according to any one of claims 16-19, wherein each peptide chain of the bispecific antibody is linked to 0, 1, 2, 3, 4, or 5 of the following structures via VHH, cysteine ​​residues in the reduced disulfide bond of the hinge region, or cysteine ​​residues in the Fc domain:

21. The antibody-drug conjugate according to any one of claims 16-20, wherein the conjugate is selected from: in, x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab is the bispecific antibody according to any one of claims 1-10.

22. The antibody-drug conjugate according to any one of claims 16-20, wherein: in, x = 1, 2, 3, 4, 5 or 6; Ab contains or is composed of the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

23. A composition comprising or consisting of one or more antibody-drug conjugates according to any one of claims 16-22; Preferably, the composition has a DAR value of 1-10.

24. A composition comprising or consisting of one or more antibody-drug conjugates of claim 22; Preferably, the DAR value of the composition is 1-8; More preferably, the composition has a DAR value of 1-5, for example 3.5-4.5, or even 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.

5.

25. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-10, a nucleic acid molecule according to claim 11, a carrier according to claim 12, a host cell according to claim 13, a conjugate according to claim 15, an antibody-drug conjugate according to any one of claims 16-22, or a composition according to any one of claims 23-24, and optionally a carrier or excipient.

26. Use of the bispecific antibody of any one of claims 1-10, the nucleic acid molecule of claim 11, the vector of claim 12, the host cell of claim 13, the conjugate of claim 15, the antibody-drug conjugate of any one of claims 16-22, the composition of any one of claims 23-24, or the pharmaceutical composition of claim 25 for the preparation of a medicament for the prevention and / or treatment in a subject of diseases related to Trop2 and / or c-Met; Preferably, the Trop2-related and / or c-Met-related disease is a tumor, such as a Trop2 and / or c-Met-positive tumor; Preferably, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer), oral squamous cell carcinoma, ovarian cancer (e.g., ovarian epithelial carcinoma), cervical cancer, and bladder cancer. Preferably, the subject is a mammal, such as a human; Preferably, the bispecific antibody, nucleic acid molecule, carrier, host cell, conjugate, antibody-drug conjugate, composition, or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents.

27. A method for detecting the presence or level of Trop2 and / or c-Met in a sample, comprising using the bispecific antibody of any one of claims 1-10 or the conjugate of claim 15; Preferably, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

28. The bispecific antibody of any one of claims 1-10 or the conjugate of claim 15; used in the preparation of a diagnostic reagent for detecting the presence or level of Trop2 and / or c-Met in a sample or for diagnosing whether a subject has a disease related to Trop2 and / or c-Met.

Images