Antibody-drug conjugates targeting b7-h3 and trop2 and uses thereof

Abstract

The present invention relates to bispecific antibodies targeting B7-H3 and TROP2, as well as antibody-drug conjugates based on the same, and compositions containing the antibody-drug conjugates. The invention also relates to therapeutic and diagnostic uses of these antibody-drug conjugates.

Description

[0001] Cross-reference to related applications

[0002] This application is based on a Chinese patent application with an application number of 202311785547.2 and a filing date of December 22, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby incorporated into this application by reference. Technical field

[0003] The present invention relates to antibodies targeting B7-H3 and TROP2, antibody-drug conjugates (ADCs) constructed based thereon, and compositions containing the antibody-drug conjugates. The present invention also relates to the therapeutic and diagnostic uses of these antibodies or ADCs. Background of the invention

[0005] Therapies using cytotoxic agents are important for treating cancer, but low-selectivity cytotoxic agents often kill normal cells, causing serious toxic side effects. Although biologic macromolecule drugs, such as antibodies or antibody fragments, have high targeting properties, their therapeutic effects on solid tumors are limited, thus restricting their applications. Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, combining the targeting effect of antibodies with the high activity of small molecule drugs (such as cytotoxic agents), and having advantages such as high efficacy and high safety.

[0006] B7-H3 is a type I transmembrane protein of the B7 family. It is not constitutively expressed in many immune cells (e.g., natural killer (NK) cells, T cells, and antigen-presenting cells (APCs)); however, its expression can be induced. In addition, the expression of B7-H3 is not limited to immune cells. B7-H3 transcripts are expressed in a variety of human tissues (including colon, heart, liver, placenta, prostate, small intestine, testis, and uterus) as well as in osteoblasts, fibroblasts, epithelial cells, and other non-lymphoid lineage cells that may indicate immune and non-immune functions (Nygren et al. Front Biosci. 3:989-93 (2011)). However, protein expression in normal tissues is usually maintained at a low level and may thus be subject to post-transcriptional regulation. B7-H3 is also expressed in a variety of human cancers, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, breast cancer, and so on.

[0007] Studies on ADCs targeting B7-H3 have been carried out (see, for example, US20200338209A1; Scribner et al., Mol Cancer Ther (2020) 19(11):2235–2244; Yamato et al., Mol Cancer Ther. 2022 Apr 1; 21(4):635–646). Among them, DS-7300a developed by Daiichi Sankyo Co., Ltd is one of the most typical blockbuster new drugs in this field, which has high potency against various cancers (Yamato et al., Mol Cancer Ther. 2022 Apr 1; 21(4):635–646). However, existing ADCs including DS-7300a still have some defects, such as the need to further improve efficacy, low endocytosis efficiency, side effects that need to be improved, uneven distribution of the product DAR value affecting drug efficacy, low DAR value and / or short half-life, etc.

[0008] TROP2, a trophoblast cell surface antigen, also known as tumor-associated calcium signal transducer 2 (TACSTD2), is overexpressed in a variety of human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, etc. (Yezhe Cheng et al., Frontiers in Oncology, 2022 Dec 23; 12:951589).

[0009] Preclinical and clinical studies have demonstrated the use of anti-TROP2 antibody-drug conjugates (ADCs) (such as Trodelvy, anti-human TROP2 antibody-SN-38 conjugate, or DS-1062a) for the treatment of cancer. Clinical results show that good therapeutic effects have been achieved in the treatment of refractory solid tumors using Trodelvy. The objective response rate (ORR) of Trodelvy reached 33% in patients with resistant triple-negative breast cancer (TNBC). One of the important mechanisms of action of Trodelvy is that SN-38 (payload) is linked through a pH-sensitive linker, and the linker can break and specifically release SN-38 in the acidic tumor microenvironment. However, because the linker of Trodelvy is not stable enough, the maleimide-mediated linker breaks through thiol exchange under physiological conditions, making the half-life of Trodelvy in serum relatively short (about 1 day). Therefore, Trodelvy may have a relatively high off-target effect. Although Ds-1062a (Dato-DXd, AstraZeneca and Daiichi Sankyo) has significant efficacy in the treatment of patients with non-mutated lung cancer and triple-negative breast cancer, there are still problems such as uneven DAR distribution affecting drug efficacy and target toxicity in normal tissues.

[0010] In addition, ADCs of monoclonal antibodies also have the problem of drug resistance caused by decreased target expression.

[0011] Therefore, there is a need in the art to develop new bispecific antibodies based on B7-H3 and TROP2, as well as ADC molecules based thereon. Preferably, such molecules have advantages such as high efficacy, high safety (including low side effects), and / or high product homogeneity. Summary of the Invention

[0013] The present invention provides an immunoconjugate comprising an antibody targeting B7-H3 and TROP2 (e.g., a bispecific antibody specifically binding B7-H3 and TROP2 of the present invention) and other payloads.

[0014] In some aspects, the immunoconjugate is an antibody-drug conjugate.

[0015] Therefore, the present invention provides a bispecific antibody-drug conjugate (ADC) targeting B7-H3 and TROP2.

[0016] In some embodiments, the ADC has one or more of the following advantages:

[0017] (1) It can target two antigens simultaneously due to the bispecific antibody contained therein.

[0018] (2) It has higher endocytosis efficiency.

[0019] (3) It has advantages such as higher affinity.

[0020] In one aspect, the present invention provides an antibody-drug conjugate having formula (I):

[0021] Ab-(L-D) p (I)

[0022] or a pharmaceutically acceptable salt or solvate thereof.

[0023] Wherein:

[0024] Ab is an antibody or a fragment thereof that specifically binds B7-H3 and TROP2 (e.g., human B7-H3 and TROP2);

[0025] L is a linker;

[0026] D is a drug, preferably an anti-tumor compound; and

[0027] p is an integer selected from 1 to 16, such as 4-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0028] Wherein Ab in formula (I) is a bispecific antibody that specifically binds to B7-H3 and TROP2, which comprises a first antigen-binding region that specifically binds to TROP2 and a second antigen-binding region that specifically binds to B7-H3, wherein the second antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein

[0029] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of said sequence, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of said sequence, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 or consists of said sequence, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7 or consists of said sequence, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8 or consists of said sequence, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of said sequence.

[0030] In some embodiments, the second antigen-binding region of the bispecific antibody that specifically binds to B7-H3 comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 4 or consists of said amino acid sequence, and / or the VL comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 10 or consists of said amino acid sequence.

[0031] In some embodiments, the second antigen-binding region of the bispecific antibody that specifically binds to B7-H3 comprises VH and VL, wherein VH and VL respectively comprise the amino acid sequences shown below or consist of said amino acid sequences: SEQ ID NO: 4 and SEQ ID NO: 10.

[0032] In some embodiments, the first antigen-binding region of the bispecific antibody that specifically binds to TROP2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein

[0033] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:19; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:20; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:21.

[0034] In some embodiments, the first antigen-binding region of the bispecific antibody that specifically binds to TROP2 comprises VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consists of said amino acid sequence, and / or the VL contains the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consists of said amino acid sequence.

[0035] In some embodiments, the first antigen-binding region of the bispecific antibody that specifically binds to TROP2 comprises VH and VL, wherein VH and VL respectively contain the amino acid sequences shown below or consist of said amino acid sequences: SEQ ID NO:16 and SEQ ID NO:22.

[0036] In some embodiments, the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different. In some embodiments, the first Fc region and the second Fc region are each a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, such as comprising the amino acid sequence SEQ ID NO:30 or 31 or an amino acid sequence having at least 90%, such as 95%, 96%, 97%, 98%, 99% or higher identity to the amino acid sequence or consisting thereof. In some embodiments, mutations that promote heterodimerization of the first Fc region and the second Fc region are introduced into the first Fc region and the second Fc region of the bispecific antibody. In some embodiments, the mutations in the Fc region are introduced based on the Knob-into-Hole technology, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region, respectively. In some embodiments, one Fc region of the bispecific antibody comprises the amino acid substitutions S354C and T366W, and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index), such as wherein S354C and Y349C form a non-natural disulfide bond.

[0037] In some embodiments, the mutations in the Fc region of the bispecific antibody are introduced based on the Innobody technology.

[0038] In some embodiments, the Fc region of the bispecific antibody further comprises mutations that reduce binding to Fcγ receptors, such as the L234A / L235A mutation.

[0039] In some embodiments, one Fc region polypeptide of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:27 or consisting thereof, while the other Fc region polypeptide comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:28 or consisting thereof.

[0040] In some embodiments, the first antigen-binding region of the bispecific antibody is a Fab fragment, and / or the second antigen-binding region is a Fab. In some embodiments, in the bispecific antibody, the Fab serving as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1 comprises

[0041] (i) comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:32 or consisting of said amino acid sequence; or

[0042] (ii) comprising the amino acid sequence of SEQ ID NO:32 or consisting of said amino acid sequence.

[0043] In some embodiments, the Fab serving as the first antigen-binding region or the second antigen-binding region in the bispecific antibody comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, the Kappa light chain constant region comprises

[0044] (i) comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:11 or consisting of said amino acid sequence; or

[0045] (ii) comprising the amino acid sequence of SEQ ID NO:11 or consisting of said amino acid sequence.

[0046] In some embodiments, in the bispecific antibody, the first antigen-binding region that specifically binds to TROP2 comprises a first Fab, and the second antigen-binding region that specifically binds to B7-H3 comprises a second Fab, wherein the first Fab is linked to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the second Fab is linked to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region).

[0047] In some embodiments, the bispecific antibody is an IgG-like antibody having a configuration as Figure 1 shown.

[0048] In some embodiments, the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds to TROP2 and a second Fab as a second antigen-binding region that specifically binds to B7-H3, wherein the bispecific antibody comprises or consists of:

[0049] Heavy chain 1: from the N-terminus to the C-terminus comprises or consists of: the heavy chain variable region of the Fab that specifically binds to TROP2 - the heavy chain constant region CH1 - the first Fc region, wherein the heavy chain constant region CH1 is connected to the N-terminus of the first Fc region at its C-terminus via or without a linker (such as a hinge region);

[0050] Light chain 1: from the N-terminus to the C-terminus comprises or consists of: the light chain variable region of the Fab that specifically binds to TROP2 - the light chain constant region;

[0051] Heavy chain 2: from the N-terminus to the C-terminus comprises or consists of: the heavy chain variable region of the Fab that specifically binds to B7-H3 - the heavy chain constant region CH1 - the second Fc region, wherein the heavy chain constant region CH1 is connected to the N-terminus of the second Fc region at its C-terminus via or without a linker (such as a hinge region);

[0052] Light chain 2: from the N-terminus to the C-terminus comprises or consists of: the light chain variable region of the Fab that specifically binds to B7-H3 - the light chain constant region,

[0053] Preferably, each domain is directly connected;

[0054] Optionally, the first Fc region comprises the mutations Y349C, T366S, L368A, and Y407V, and the second Fc region comprises the mutations S354C and T366W, and vice versa; optionally, the first Fc region and the second Fc region further comprise the L234A / L235A mutations, respectively.

[0055] In some embodiments, the heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; the light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; the heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence.

[0056] In some embodiments, the anti-tumor compound is a cytotoxic agent, such as camptothecin compounds, auristatin compounds, maytansine compounds, taxanes, anthracyclines, vinca alkaloids, MEK inhibitors or KSP inhibitors.

[0057] In some embodiments, D is as shown in formula (D-1):

[0058]

[0059] wherein R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0060] R 2 is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and -SR 4 ; R 3 is selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 together form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0061] R 4 is selected from H or C1-C4 alkyl.

[0062] It should be understood that the wavy lines in the structures in the text indicate the connection positions of the structures. For example, the wavy line in formula (D-1) indicates the connection position of D-1 and L.

[0063] In some embodiments, R 1 is H, R 2 is a C1-C6 alkyl group, and R 3 is -F.

[0064] In some embodiments, D is as shown in formula (D-2):

[0065]

[0066] wherein R 1 , R 2 and R 3 are as defined above.

[0067] In some embodiments, D is as shown in formula (D-3) or formula (D-4):

[0068]

[0069] In some embodiments, -L- is the following structure:

[0070] -Z-E-NH-CH2-Q-L 2 -L 1 -

[0071] where Z is connected to Ab, and L 1 is connected to D;

[0072] Z is selected from where m is an integer from 1 to 10; the carbonyl group at the right end of Z is covalently connected to E;

[0073] E is a peptide residue containing 2-10 amino acids, wherein the peptide residue is optionally substituted by one or more polyol groups, and the N-terminus of the peptide residue is covalently connected to Z;

[0074] Q is -O- or -S-;

[0075] L 1 is absent or -(C1-C 10 alkylene)-;

[0076] L 2 is absent, -N(R 5 )(C=O)-(C1-C 10 alkylene)-* or -(C=O)N(R 5 )(C1-C 10 alkylene)-*; where * indicates that the terminus is covalently connected to Q; and

[0077] R 5 is H or a C1-C6 alkyl group.

[0078] In some embodiments, E is a peptide residue consisting of 2, 3, or 4 amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein said glutamine or glutamic acid is optionally substituted with 1 polyol group; and

[0079] -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10 alkylene)-*, where * indicates that the terminus is covalently linked to Q;

[0080] R 5 is H or a C1-C6 alkyl group.

[0081] In some embodiments, the polyol group is

[0082] In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala- or wherein R 6 is H or a C1-C6 alkyl group, where these E groups are covalently linked to Z through the left N-terminus; and

[0083] -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0084] In some embodiments, -Z-E-NH-CH2-Q-L 2 -L 1 - has the following structure

[0085]

[0086] whose right end is linked to D.

[0087] In some embodiments, the antibody-drug conjugate is as shown in the following formula

[0088] Ab’-(S-L-D) p (I’)

[0089] where Ab’ is as defined above for Ab, and L, D, and p are as defined above.

[0090] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR value of 5-11, such as 7.5-8.5.

[0091] In some embodiments, the antibody-drug conjugate is as shown in the following structure:

[0092]

[0093] wherein Ab is the bispecific antibody of the present invention; q is as defined for p in formula (I) above,

[0094] Preferably, the antibody-drug conjugate has an average DAR of 5-11 or 7.5-8.5.

[0095] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

[0096] In another aspect, the present invention provides a drug combination comprising the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0097] In another aspect, the present invention provides a method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described above, or the pharmaceutical composition as described above, or the drug combination as described above.

[0098] In some embodiments, the tumor is cancer, preferably, the cancer has elevated levels (e.g., at the nucleic acid or protein level) of B7-H3 and / or TROP2, such as compared to the corresponding tissue of a healthy individual or to healthy tissue adjacent to the cancer tissue of the patient.

[0099] In some embodiments, the cancer is selected from lung cancer (including non-small cell lung cancer and small cell lung cancer), colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck cancer, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.

[0100] In some embodiments, the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents. Preferably, the treatment modality includes radiotherapy or surgery, or the therapeutic agent includes a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, small molecule drugs, or an immunomodulator.

[0101] The antibody-drug conjugate of the present invention has the following advantages:

[0102] (1) Binds to target cells expressing human B7-H3 and / or TROP2 with high affinity.

[0103] (2) Can enter cells through endocytosis to kill target cells.

[0104] (3) Has high endocytosis efficiency.

[0105] (4) Has a significant bystander effect.

[0106] (5) Has high anti-tumor efficacy, has a stronger killing effect on tumor cells, has a stronger inhibitory effect on tumor growth, especially tumors with high expression of B7-H3 and / or TROP2; has significantly improved or even unexpectedly high anti-tumor activity.

[0107] (6) Has a DAR value of up to about 8, and the product is more homogeneous.

[0108] (7) Has low toxicity.

[0109] (8) Has good stability, and the linker-payload used in the present invention has high stability.

[0110] (9) Has good drug-likeness. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 Shows a schematic diagram of the structure of the bispecific antibody HZ5C2.9 / hRS7.13.

[0112] Figure 2 Shows the binding experiment results of the bispecific antibody HZ5C2.9 / hRS7.13 and the parental antibody to NCI-H358 cells.

[0113] Figure 3 Shows the endocytosis experiment results of the bispecific antibody HZ5C2.9 / hRS7.13 and the parental antibody.

[0114] Figure 4 Shows the binding experiment results of the bispecific ADC molecule and the parental antibody ADC molecule to cells.

[0115] Figure 5Shows the in vitro killing effects of bispecific ADC molecules and parental antibody ADC molecules on multiple tumor cell lines.

[0116] Figure 6A Shows the tumor suppression effects of bispecific ADC molecules and parental antibody ADC molecules in the NCI-H358 mouse xenograft tumor model.

[0117] Figure 6B Shows the changes in body weight of mice in the tumor suppression experiment using the NCI-H358 mouse xenograft tumor model.

[0118] Figure 7A Shows the tumor suppression effects of bispecific ADC molecules and parental antibody ADC molecules in the HCC1806 mouse xenograft tumor model.

[0119] Figure 7B Shows the changes in body weight of mice in the tumor suppression experiment using the HCC1806 mouse xenograft tumor model.

[0120] Figure 8 Shows a schematic diagram of the bispecific ADC molecule. DETAILED DESCRIPTION OF THE INVENTION

[0122] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0123] I. DEFINITIONS

[0124] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural, and vice versa, where appropriate. It is to be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0125] The term "about", when used in conjunction with a numerical value, means to cover a numerical value within a range having a lower limit that is 5% less (e.g., 4%, 3%, 2%, or 1%) than the specified numerical value and an upper limit that is 5% greater (e.g., 4%, 3%, 2%, or 1%) than the specified numerical value.

[0126] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0127] As used herein, the term "comprising" or "including" means including the stated element, integer or step, but not excluding any other element, integer or step. In this text, when the term "comprising" or "including" is used, unless otherwise specified, the situation composed of the stated element, integer or step combination is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover an antibody variable region composed of this specific sequence.

[0128] Unless otherwise indicated, the terms "B7-H3", "B7H3" and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to the B7 / CD28 superfamily members and is similar in sequence to the extracellular domain of PD-L1. B7-H3 has 316 amino acids, including a putative signal peptide consisting of 28 amino acids, an extracellular region consisting of 217 amino acids, a transmembrane region and a cytoplasmic domain consisting of 45 amino acids, and its molecular weight is about 45-66 kDa. In humans, due to exon duplication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3), or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 has about 90% homology with its human counterpart. In some embodiments of the present invention, B7-H3 is human B7-H3. In some embodiments, B7-H3 is the protein under UniProt database accession number Q5ZPR3.

[0129] The term "anti-B7-H3 antibody", "anti-B7-H3", "B7-H3 antibody" or "antibody against B7-H3" as used herein refers to an antibody that can bind to the B7-H3 protein with sufficient affinity. The antibody can be used as a diagnostic agent and / or therapeutic agent targeting B7-H3, or for constructing immunoconjugates, such as antibody-drug conjugates.

[0130] The term "anti-TROP2 antibody", "anti-TROP2", "TROP2 antibody" or "antibody against TROP2" as used herein refers to an antibody that can bind to the TROP2 protein with sufficient affinity. The antibody can be used as a diagnostic agent and / or therapeutic agent targeting TROP2, or for constructing immunoconjugates, such as antibody-drug conjugates.

[0131] In some embodiments of the present invention, TROP2 is human TROP2. In some embodiments, TROP2 is the protein under UniProt database accession number P09758.

[0132] As used herein, when referring to "first" and "second", it is only for distinguishing two domains or two chains, and does not indicate the positions of the two domains in any way.

[0133] General information on the amino acid sequences and nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0134] As used herein, the amino acid positions of the entire variable regions of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as "Kabat numbering".

[0135] As used herein, when referring to the amino acid positions in domains of an antibody other than the variable regions (e.g., constant regions, e.g., Fc regions), they are numbered according to the EU numbering system described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as "EU numbering". When position numbers and / or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, which is known to those skilled in the art.

[0136] The term "antibody" is used herein in the broadest sense to refer to a protein containing an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.

[0137] The terms "whole antibody", "full-length antibody", "complete antibody", and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3 (and optionally CH4). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH region and the VL region can be further subdivided into hypervariable regions (complementary determining regions (CDRs)) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions.

[0138] "Half antibody" or "halfmer" refers to a monovalent antigen-binding polypeptide. In some embodiments, the half antibody or halfmer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, the half antibody or halfmer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, the half antibody or halfmer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, the half antibody binds to TROP2 and does not bind to B7-H3. In some specific embodiments, the half antibody binds to B7-H3 and does not bind to TROP2. Those skilled in the art will readily understand that the half antibody can have an antigen-binding domain consisting of a single variable domain (e.g., derived from Camelidae).

[0139] In the present invention, the antibody constant region or antibody constant domain, including CH1, CL, and Fc domains, as well as the CH2, CH3, and optional CH4 domains constituting the Fc domain, can be selected according to the expected function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG, or IgM region, particularly the immunoglobulin constant domain of human IgG, such as the constant domains of human IgG1, IgG2, IgG3, or IgG4, preferably the constant domain of human IgG1. For another example, the Fab fragment of the antibody can comprise the CH1 and CL constant regions from IgG1. For another example, the Fc region of the antibody can comprise the CH2 and CH3 domains from IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0140] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, namely the CH2 domain, the CH3 domain, and optionally the CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from antibodies of the IgG, IgA, and IgD classes; or contains the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from antibodies of the IgM and IgE classes. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimeric Fc, and two different Fc regions heterodimerize to form a heterodimeric Fc. As used herein, the terms "Fc region", "Fc portion", and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and the light chain variable region VL of an immunoglobulin, nor the heavy chain constant region CH1 and the light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxyl terminus of the heavy chain.

[0141] In one embodiment, a human IgG1 Fc region polypeptide (including a portion of the hinge region) comprises or consists of the following amino acid sequence:

[0142] DKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:30).

[0143] In one embodiment, the human IgG1 Fc region polypeptide (including a partial hinge region) comprises the following amino acid sequence or consists thereof, wherein E is at position 356 and M is at position 358:

[0144] DKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:31).

[0145] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or part of the human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass.

[0146] As used herein, the "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be linked at its N-terminus or C-terminus to an antigen-binding domain (e.g., the heavy and / or light chain variable regions of an antibody that can bind to a target molecule or an antigen-binding fragment of an antibody, or a soluble portion of a ligand or receptor that can bind to a target molecule) for constructing a multispecific antibody such as a bispecific antibody.

[0147] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises

[0148] the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:32) or consists thereof.

[0149] The term "antibody fragment" includes a portion of a full-length antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment. The term "antigen-binding fragment" of an antibody is a molecule distinct from the full-length antibody that comprises a portion of the full-length antibody but that binds the antigen of the full-length antibody or competes with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to the antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of a full-length antibody. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), or nanobody.

[0150] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains and comprising the variable region of the immunoglobulin heavy chain (VH), the constant domain of the heavy chain (CH1), the variable region of the light chain (VL), and the constant domain of the light chain (CL), wherein one polypeptide chain comprises, from the N-terminus to the C-terminus, VH and one constant region selected from CH1 and CL, and the other polypeptide chain comprises, from the N-terminus to the C-terminus, VL and the other constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen-binding site or antigen-binding region. In the present context, the Fab polypeptide chain comprising the heavy-chain constant region CH1 is also referred to as the "Fab heavy chain"; correspondingly, the Fab polypeptide chain comprising the light-chain constant region CL is also referred to as the "Fab light chain".

[0151] "Complementary determining region" or "CDR region" or "CDR" is a region in the variable domain of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen - contacting residues ("antigen - contact points"). CDRs are mainly responsible for binding to epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N - terminus. The CDRs located within the variable region of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable region of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a variable light - chain or variable heavy - chain region, the precise amino - acid sequence boundaries of each CDR can be determined using any one or a combination of many well - known antibody CDR assignment systems, including, for example: Chothia, based on the three - dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877 - 883, Al - Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927 - 948 (1997)), Kabat, based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (www.imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above - mentioned ways or a combination thereof. CDRs can also be determined based on having the same Kabat - numbered positions as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0152] In one embodiment, the CDRs of the antibodies of the present invention have their boundaries determined by Kabat rules, or by AbM rules, or by a combination - determining rule thereof.

[0153] In one embodiment of the present invention, the HCDR1 of VH in the antigen-binding region that binds to B7-H3 in the present invention is determined by the AbM rule, the HCDR2 and HCDR3 are determined by the Kabat rule, and the LCDRs of VL are determined by the Kabat rule respectively.

[0154] In one embodiment of the present invention, the HCDR1 of VH in the antigen-binding region that binds to TROP2 in the present invention is determined by the AbM rule, the HCDR2 and HCDR3 are determined by the Kabat rule, and the LCDRs of VL are determined by the Kabat rule respectively.

[0155] The term "hinge region" refers to the portion of the antibody heavy chain polypeptide that, in the wild-type antibody heavy chain, links the CH1 region and the CH2 region. For example, the IgG1 hinge region, such as the sequence from D221 to P230 according to EU numbering. The hinge regions of other IgG subclasses can be determined by aligning with the hinge region cysteine residues of the IgG1 subclass sequence. In some embodiments, CH1 can include a portion of the hinge region. In some embodiments, the Fc region can include a portion of the hinge region.

[0156] Amino acid mutations are represented by (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is in the Fc region, "T366W" means that T at EU numbering position 366 is replaced by W. When referring to a combination of mutations, the combined mutations are connected by "and" or " / ". "L234A / L235A" means that both mutations L234A and L235A are included. It should be noted that when describing mutations, the specific position also covers the corresponding amino acid positions on other polypeptide chains.

[0157] For a polypeptide sequence, "conservative alteration" includes substitution, deletion, or addition to the polypeptide sequence, but does not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of one amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M). In some embodiments, the term "conservative sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen-binding characteristics of the antibody molecules of the invention that contain the amino acid sequence. For example, conservative modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100 - 110% or higher binding affinity for the target antigen relative to the parental antibody.

[0158] The term "target" refers to the entity to which a binding molecule is directed. The target can be an antigen, or it can be a ligand or receptor. The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the invention relates to a target-binding region derived from an antibody, "target" and "antigen" can be used interchangeably.

[0159] The term "antigen-binding region" as used herein refers to any part of an antibody or an antigen-binding fragment thereof, such as a multispecific antibody or a bispecific antibody, that binds to a specific target or antigen. The antigen-binding region can be, for example, an antibody or an immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody, and may bind or not bind its antigen / epitope as a separate entity.

[0160] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of the at least two antigen-binding regions binding to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is such a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0161] As used herein, the term "bispecific antibody" encompasses an antigen-binding domain that specifically binds to two antigens or two epitopes. Unless otherwise specified, the order of the antigens to which the bispecific antibody binds in the listed bispecific antibody names is arbitrary. That is, in some embodiments, the terms "anti-TROP2 / B7-H3 bispecific antibody" and "anti-B7-H3 / TROP2 bispecific antibody" can be used interchangeably. In some embodiments, a bispecific antibody comprises two half-antibodies, each of which comprises a single heavy-chain variable region and optionally at least a portion of the heavy-chain constant region and a single light-chain variable region and optionally at least a portion of the light-chain constant region. In some embodiments, a bispecific antibody comprises two half-antibodies, each of which comprises a single heavy-chain variable region and a single light-chain variable region and does not comprise more than one single heavy-chain variable region and does not comprise more than one single light-chain variable region. In some embodiments, a bispecific antibody comprises two half-antibodies, each of which comprises a single heavy-chain variable region and a single light-chain variable region, and wherein the first half-antibody binds to the first antigen / epitope and does not bind to the second antigen and the second half-antibody binds to the second antigen / epitope and does not bind to the first antigen.

[0162] When referring to the "first antigen-binding region" in a multispecific antibody or a bispecific antibody, it refers to the binding region that binds to the first antigen, without intending to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or a bispecific antibody may contain 1 or more than 1 first antigen-binding regions. For example, a bispecific antibody comprises a first antigen-binding region and a second antigen-binding region, but may contain 1 or more than 1 first antigen-binding regions and 1 or more than 1 second antigen-binding regions.

[0163] When referring to "a target or antigen-binding region is derived from an antibody", it means that the binding domain constituting the target / antigen-binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen. For example, a fragment of the antigen-binding region that specifically binds to the antigen, such as Fab, is or is derived from the corresponding fragment of the antibody, such as Fab, or the heavy-chain variable region and / or light-chain variable region of the antigen-binding region is or is derived from the heavy-chain variable region and / or light-chain variable region of the antibody, or 1, 2, 3, 4, 5, or 6 CDRs of the antigen-binding region are the CDRs of the antibody.

[0164] The term "derived from" means that the fragment in the antigen-binding region is substantially the same as the fragment of the antibody from which it is derived, but may have mutations at one or more sites, such as substitutions, deletions or additions. In one specific embodiment, the mutations are not in the CDRs of the antibody.

[0165] The multispecific antibodies of the present invention may comprise linkers. As used herein, the term "linker" refers to any molecule that enables the direct connection of different parts of a multispecific antibody. Examples of linkers that establish covalent linkages between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol or polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to a sequence of amino acids, wherein the sequence joins together the amino acid sequences of the various parts of the multispecific antibody. Preferably, the peptide linker has a length sufficient to join two entities in such a way that they maintain their conformation relative to each other such that the desired activity is not impeded. Peptide linkers may or may not predominantly comprise the following amino acid residues: Gly, Ser, Ala or Thr.

[0166] The term "effector function" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen presentation cell uptake of antigen, downregulation of cell surface receptors (e.g., B cell receptors) and B cell activation.

[0167] The term "…valent" antibody refers to the number of antigen-binding sites present in an antibody molecule. "Bivalent", "trivalent" and "tetravalent" antibodies refer to antibody molecules having 2, 3 and 4 antigen-binding sites, respectively.

[0168] The "knob-into-hole (KIH)" mutation, or "knob-into-hole" mutation, or "KIH" mutation is used herein to refer to the introduction of mutations in a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-into-hole" technique, to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. As is known in the art, the "knob-into-hole" technique can engineer the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule. Generally, the technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "cavity / hole" at the interface of the other chain to which it is to be paired, such that the protrusion can be placed in the cavity. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to which it is to be paired. The protrusion can be constructed by replacing small amino acid side chains at the interface of the CH3 domain of the heavy chain constant domain from one chain with larger side chains (such as tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (such as alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to which it is to be paired. Another alternative interface is the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, and the correct heterodimerization between the two chains of the Fab fragment is promoted by constructing a protrusion-cavity interaction. In some embodiments, the KIH mutation also comprises cysteine mutations in the two CH3 domains to form non-native disulfide bonds.

[0169] As used herein, the terms "bind" or "specifically bind" mean that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or by conventional binding assays known in the art, such as by radioimmunoassay (RIA) or by biolayer interferometry or MSD assay or surface plasmon resonance (SPR) assay.

[0170] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between the members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (Kdis and Kon, respectively). Affinity can be measured by common methods known in the art. A specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0171] The sequence identity between sequences is calculated as follows. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0172] As used herein, an "immunoconjugate" refers to a conjugate in which a payload is linked to an antibody or an antigen-binding fragment thereof through a linker, such that the antibody or its antigen-binding fragment can serve as a carrier to target the transport of the payload to a target site. The term "payload" refers to the active portion conjugated to an antibody or antibody fragment of the present invention and can include any moiety for attaching to the antibody or antibody fragment. In some embodiments, the payload can be a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide, etc. In some embodiments, immunoconjugates encompass antibody-drug conjugates (ADCs), antibody-immunostimulatory conjugate drugs (ISACs), antibody-oligonucleotide conjugates (AOCs), antibody-polypeptide conjugate drugs (APCs), antibody-radionuclide conjugate drugs (RDCs), or antibody-degradation conjugate drugs (ADeCs), etc. Payloads or active moieties suitable for linking to an antibody include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonists (TLRs) such as ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonists such as ISAC drug XMT-2056, Treg cell modulating ISAC drug ADCT-301, etc.), immunomodulators, therapeutic oligonucleotides (siRNA, PMO, etc.), or radionuclides, etc. In some embodiments, the immunoconjugate of the present invention is an antibody-drug conjugate, i.e., an ADC.

[0173] As used herein, an "antibody-drug conjugate (ADC)" refers to a compound / molecule obtained by linking an antibody to a drug (e.g., a small molecule drug) through a linker.

[0174] The term "linker" refers to a structural moiety that links a drug (e.g., a small molecule drug) to the antibody moiety. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being linked to the antibody or its antigen-binding fragment.

[0175] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (e.g., a small molecule drug), to a linker.

[0176] The term "site-specific conjugation" as used herein refers to the conjugation of a drug specifically to a specific site of an antibody through a linker.

[0177] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl preferably contains 1-16 carbon atoms, such as 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms or 1-4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0178] The term "alkylene" refers to an alkyl group as defined above, but it is divalent, i.e., having two single bonds connected to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)- or -CH2CH(-CH3)-.

[0179] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group containing 2-16 carbon atoms and containing at least one double bond and no triple bond. The alkenyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms or 2-4 carbon atoms. Representative examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0180] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group containing 2-16 carbon atoms and containing at least one triple bond. The alkynyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms or 2-4 carbon atoms. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.

[0181] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br) and iodine (-I).

[0182] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. The haloalkyl can preferably be a monohaloalkyl, dihaloalkyl or polyhaloalkyl (including perhaloalkyl). The monohaloalkyl group can contain one iodine, bromine, chlorine or fluorine in the alkyl. The dihaloalkyl and polyhaloalkyl groups can contain two or more identical halogen atoms or a combination of different halo groups in the alkyl. Preferably, the polyhaloalkyl contains at most 12, 10, 8, 6, 4, 3 or 2 halogen groups. Non-limiting examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0183] The term "haloalkenyl" refers to an alkenyl group as defined herein that is substituted with one or more halogen groups as defined herein. The term "haloalkynyl" refers to an alkynyl group as defined herein that is substituted with one or more halogen groups as defined herein. The meaning of "halo" as defined for "haloalkyl" is applicable to "haloalkenyl" and "haloalkynyl".

[0184] The term "polyol group" refers to an alkyl group as defined above that contains multiple (e.g., 2 - 10, such as 3, 4, 5, 6, 7 or 8) hydroxyl groups and optionally contains one or more (e.g., 2, 3 or 4) other groups (e.g., amino, carbonyl). Non-limiting examples of "polyol group" include, for example

[0185] The term "amino acid" refers to naturally occurring and synthetic amino acids. The amino acid can be an L or D isomer. The notations of conventional amino acids involved herein follow conventional usage. See, for example, Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in the present disclosure, amino acids are generally represented by the single-letter and three-letter abbreviations well-known in the art. For example, glycine can be represented by Gly, alanine by Ala, valine by Val, glutamine by Gln, glutamate by Glu, phenylalanine by Phe and leucine by Leu.

[0186] The term "optional" or "optionally": means that the subsequently described event or circumstance may or may not occur, and this description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted", the group or structure may be substituted or may not be substituted.

[0187] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention and that is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist in their pharmaceutically acceptable salt forms, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugate in the present invention with an organic or inorganic acid, and the organic or inorganic acid includes but is not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. A pharmaceutically acceptable non-toxic base addition salt refers to a salt formed by the ADC conjugate in the present invention with an organic or inorganic base, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reaction with an organic base containing an N group.

[0188] The term "solvate" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate in the present invention. Solvents that form solvates include but are not limited to water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0189] Where there is no contradiction in the context, the terms "pharmaceutically acceptable" and "medicinal" are used interchangeably herein.

[0190] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) conjugated to the Ab moiety in an ADC molecule to the Ab moiety. In some embodiments described herein, the DAR can be determined by p in Formula I. For example, the DAR can be from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug moiety (D) conjugated to the Ab moiety in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and this DAR is referred to as the average DAR in the text. In some embodiments, the average DAR value of the conjugate of the present invention is from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8., 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and the range with two of these values as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules, which contains ADC molecules with the same and / or different DARs.

[0191] The term "therapeutic agent" as described herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0192] The term "cytotoxic agent" as used in the present invention refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.

[0193] "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer or immune system diseases.

[0194] The term "drug" refers to an organic compound capable of regulating biological processes, particularly of altering or preventing a pathological process.

[0195] The term "prodrug" refers to an active or inactive compound that has been chemically modified and, when administered to an individual, is converted into an active drug through physiological actions in the body (such as hydrolysis, metabolism, etc.). Techniques related to the manufacture and use of prodrugs are well known to those skilled in the art.

[0196] "Angiogenesis inhibitor" refers to a compound that blocks or in some way interferes with blood vessel development. An angiogenesis inhibitor can be, for example, a small molecule or an antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.

[0197] The term "small molecule drug" refers to an organic compound of low molecular weight capable of regulating biological processes, particularly of altering or preventing a pathological process. "Small molecule" is defined as a molecule having a molecular weight less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 D. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0198] The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates (such as activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressive agents or immunostimulatory agents. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0199] "Antitumor compound" refers to a pharmaceutically active compound that has an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773, such as camptothecin compounds exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE), maytansine compounds such as small molecule microtubule inhibitor DM1, taxanes such as paclitaxel or docetaxel, anthracyclines, epothilones, mitomycin, combretastatin, vinca alkaloids, calicheamicin, doxamicin, microtubulysin, amanitin, bleomycin, MEK inhibitors, KSP inhibitors, etc. It should be understood that antitumor compounds can be substituted by isotopes including but not limited to, for example, deuterium, tritium, etc. For example, after substitution with deuterium, when a carbon-deuterium bond replaces a carbon-hydrogen bond, since the former is more stable than the latter, this substitution can directly affect properties such as the absorption, distribution, metabolism, and excretion of certain drugs, thereby improving the efficacy, safety, and tolerance of the drugs. Therefore, the "antitumor compounds" of the present application can cover compounds substituted with deuterium.

[0200] "Substituted with deuterium" means that the hydrogen in a molecule is replaced by deuterium, for example, 1 or more hydrogens, such as 1 - 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) hydrogens are replaced by deuterium.

[0201] The term "effective amount" refers to such an amount or dose of the antibody or ADC molecule or composition or combination of the present invention that, when administered to a patient in a single or multiple doses, produces an expected effect in a patient in need of treatment or prevention. Depending on the expected effect, it can include "therapeutically effective amount" and "prophylactically effective amount".

[0202] "Therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dose and for the required period of time. A therapeutically effective amount is also such an amount that any toxic or harmful effects of the antibody or ADC molecule or composition or combination are less than the therapeutic beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (such as tumor volume) by at least about 20%, more preferably at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%.

[0203] "Prophylactically effective amount" refers to an amount that effectively achieves the desired prophylactic result at the required dose and for the required period of time. Generally, since prophylactic doses are used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0204] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny having the same functionality or biological activity as screened or selected in the originally transformed cells are included herein.

[0205] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent, such as a polynucleotide probe or antibody, and facilitates the detection of the reagent to which it is conjugated or fused. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to encompass both the direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody and the indirect labeling of a probe or antibody by reaction with another directly labeled reagent.

[0206] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0207] An "isolated" antibody or other molecule is an antibody or molecule that has been separated from the components of its natural environment or the environment in which it is expressed. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0208] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0209] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0210] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in a mammal that is typically characterized by unregulated cell growth. Cancer can be in an early, intermediate, or late stage or metastatic cancer. Cancers suitable for treatment by the molecules of the present invention include, but are not limited to, for example, lung cancer (including non-small cell lung cancer and small cell lung cancer), colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, or pancreatic cancer, including metastatic forms of these cancers.

[0211] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Tumor" encompasses solid tumors and hematological tumors as well as metastatic lesions. The terms "cancer", "cancerous", and "tumor" are not mutually exclusive when referred to herein.

[0212] "Tumor immune escape" refers to the process by which a tumor evades immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "treated" when such escape is attenuated, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0213] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, carriers, or stabilizers, etc., that are administered together with an active substance.

[0214] The term "pharmaceutical composition" refers to a composition that exists in a form that permits the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0215] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibody molecule or ADC molecule of the present invention, and (ii) other therapeutic agents) are administered to the patient in sequence as separate entities simultaneously, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the antibody molecule or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at a level not exceeding that when they are used alone. The term "fixed combination" means that two or more active agents are administered to the patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in a separate formulation, which can be the same or different.

[0216] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatment methods (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a plurality of or in separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0217] As used herein, "treat," ...

[0218] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0219] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid molecule linked thereto. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0220] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as from a fresh, frozen, and / or preserved organ or tissue sample, biopsy sample, or aspirate sample; blood or any blood component; body fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development. A tissue sample may contain compounds that are not naturally commingled with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0221] Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchoalveolar lavage fluids, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

[0222] II. Bispecific Antibodies

[0223] One aspect of the present invention provides a multispecific antibody that specifically binds to TROP2 and B7-H3.

[0224] In some embodiments, the multispecific antibody of the present invention comprises a first binding specificity for TROP2 and a second binding specificity for B7-H3, and optionally other binding specificities.

[0225] In some embodiments, the multispecific antibody is a bispecific antibody.

[0226] Accordingly, the present invention relates to a bispecific antibody that specifically binds to TROP2 and B7-H3. Thus, one aspect of the present invention relates to a multispecific antibody that comprises

[0227] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TROP2, and / or the second antigen-binding region specifically binds to B7-H3, and optionally other antigen-binding regions.

[0228] Thus, one aspect of the present invention relates to a bispecific antibody that comprises

[0229] A first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TROP2, and / or the second antigen-binding region specifically binds to B7-H3.

[0230] The bispecific antibodies of the present invention can be prepared using bispecific antibody formats or techniques known in the art. Specific exemplary bispecific formats that can be used in the context of the present invention are described, for example, in Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8): 1-24. In one embodiment, the bispecific antibody format comprises an IgG-like antibody (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy and light chains of each Fab can be from separate monoclonal antibodies. In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies that comprise a Fab fragment specifically binding to TROP2 as one antigen-binding region and a Fab fragment specifically binding to B7-H3 as the other antigen-binding region.

[0231] The components of the multispecific antibodies of the present invention, such as bispecific antibodies, are described in detail below. Those skilled in the art will understand that any combination of any technical features of these components is within the scope of the present invention, unless the context clearly indicates otherwise. And those skilled in the art will understand that the antibodies of the present invention (including any form of antibody) can comprise any such combination, unless the context clearly indicates otherwise.

[0232] The antigen-binding region of II-1 that specifically binds to TROP2

[0233] In some embodiments, the first antigen-binding region of the anti-TROP2 / B7-H3 bispecific antibody suitable for the present invention can comprise or consist of an anti-TROP2 antibody or an antigen-binding fragment thereof (such as the TROP2 antibody disclosed in US9849176B2, which is incorporated herein by reference in its entirety, such as the Sacituzumab hRS7 monoclonal antibody), as long as it can specifically bind to TROP2, including but not limited to, for example, a full-length antibody specifically binding to TROP2, a half-antibody, a Fab, a Fab’, a Fab’-SH, an Fv, a single-chain antibody (such as scFv), a (Fab’)2, a single-domain antibody such as a VHH, a dAb (domain antibody), a heavy-chain antibody, or a linear antibody, etc.

[0234] In some embodiments, the antigen-binding region that specifically binds to TROP2 is derived from an antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody.

[0235] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody.

[0236] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 heavy chain variable region CDRs of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody, namely HCDR1, HCDR2, and HCDR3.

[0237] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 light chain variable region CDRs of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody, namely LCDR1, LCDR2, and LCDR3.

[0238] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody.

[0239] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises the heavy chain variable region and / or the light chain variable region of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody.

[0240] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises the Fab of a known antibody that specifically binds to TROP2, such as the TROP2 antibody disclosed in US9849176B2, such as the Sacituzumab hRS7 monoclonal antibody.

[0241] In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises three complementarity-determining regions (HCDRs) from the heavy-chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises three complementarity-determining regions (LCDRs) from the light-chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises three complementarity-determining regions (HCDRs) from the heavy-chain variable region and three complementarity-determining regions (LCDRs) from the light-chain variable region.

[0242] In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a heavy-chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a light-chain variable region (VL). In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). In some embodiments, the heavy-chain variable region comprises three complementarity-determining regions (CDRs) from the heavy-chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light-chain variable region comprises three complementarity-determining regions (CDRs) from the light-chain variable region, LCDR1, LCDR2, and LCDR3.

[0243] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and / or VL comprised by the antigen-binding region that specifically binds to TROP2 of the present invention are respectively as defined herein.

[0244] In some embodiments, the heavy-chain variable region of the antigen-binding region that specifically binds to TROP2 of the present invention

[0245] (i) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:16 or consists of said amino acid sequence; or

[0246] (ii) comprises the amino acid sequence of SEQ ID NO:16 or consists of said amino acid sequence; or

[0247] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:16 or consists of said amino acid sequence, preferably, said amino acid alterations do not occur in the CDR regions.

[0248] In some embodiments, the light chain variable region of the antigen-binding region that specifically binds to TROP2 according to the present invention

[0249] (i) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:22 or consists of said amino acid sequence; or

[0250] (ii) comprises the amino acid sequence of SEQ ID NO:22 or consists of said amino acid sequence; or

[0251] (iii) comprises an amino acid sequence having 1 or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:22 or consists of said amino acid sequence, and preferably, said amino acid alterations do not occur in the CDR regions.

[0252] In some embodiments, the three complementarity-determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to TROP2 according to the present invention, HCDR1, HCDR2, and HCDR3 are selected from

[0253] (i) the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:16, or

[0254] (ii) a sequence that collectively contains at least one and not more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in said three HCDR regions relative to the sequence in (i),

[0255] wherein the HCDRs can be determined according to any CDR-determining scheme, for example, determined according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0256] For example, said HCDR1 is determined by the Abm scheme, and said HCDR2 and HCDR3 are determined by the Kabat scheme.

[0257] In some embodiments, the three complementarity-determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to TROP2 according to the present invention, LCDR1, LCDR2, and LCDR3 are selected from

[0258] (i) the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:22, or

[0259] (ii) Sequences that, relative to the sequence in (i), contain a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions

[0260] wherein the LCDRs can be determined respectively according to any scheme for determining CDRs, for example, determined respectively according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0261] For example, the LCDR1-3 are determined respectively by the Kabat scheme.

[0262] In some embodiments, the antigen-binding region that specifically binds to TROP2 according to the present invention comprises three complementarity-determining regions (HCDRs) contained in the heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 16 and three complementarity-determining regions (LCDRs) contained in the light-chain variable region consisting of the amino acid sequence of SEQ ID NO: 22;

[0263] wherein the HCDRs and LCDRs can be determined respectively according to any scheme for determining CDRs, for example, determined respectively according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0264] For example, the HCDR1 adopts the Abm scheme, and the HCDR2, HCDR3, and LCDR1-3 are determined respectively by the Kabat scheme.

[0265] In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence, or the HCDR1 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 13.

[0266] In some embodiments, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, or consists of the amino acid sequence, or the HCDR2 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 14.

[0267] In some embodiments, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, or consists of the amino acid sequence, or the HCDR3 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 15.

[0268] In some embodiments, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:19, or the LCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:19.

[0269] In some embodiments, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:20, or the LCDR2 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:20.

[0270] In some embodiments, the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or the LCDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:21.

[0271] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0272] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0273] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:13; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:14; HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:15; LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:19; LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:20; and LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:21.

[0274] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, and HCDR3 as shown in SEQ ID NO:15; LCDR1 as shown in SEQ ID NO:19, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21.

[0275] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises VH and VL, wherein

[0276] the VH comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consisting of the amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consisting of the amino acid sequence.

[0277] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention comprises VH and VL, wherein VH and VL respectively comprise the amino acid sequences shown below or consist of the amino acid sequences: SEQ ID NO:16 and SEQ ID NO:22.

[0278] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to TROP2 of the present invention is an anti-TROP2 Fab.

[0279] The antigen-binding region of II-2 that specifically binds to B7-H3

[0280] In some embodiments, the second antigen-binding region of the anti-TROP2 / B7-H3 bispecific antibody applicable to the present invention may comprise an antibody against B7-H3 or an antigen-binding fragment thereof or consist of the same, as long as it can specifically bind to B7-H3, including but not limited to, for example, a full-length antibody, half antibody, Fab, Fab’, Fab’-SH, Fv, single-chain antibody (such as scFv), (Fab’)2, single-domain antibody such as VHH, dAb (domain antibody), heavy-chain antibody or linear antibody that specifically binds to B7-H3.

[0281] In some embodiments, the antigen-binding region that specifically binds B7-H3 comprises three complementarity-determining regions (HCDRs) from the heavy-chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds B7-H3 comprises three complementarity-determining regions (LCDRs) from the light-chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds B7-H3 comprises three complementarity-determining regions (HCDRs) from the heavy-chain variable region and three complementarity-determining regions (LCDRs) from the light-chain variable region.

[0282] In some aspects, the antigen-binding region that specifically binds B7-H3 comprises a heavy-chain variable region (VH). In some aspects, the antigen-binding region that specifically binds B7-H3 comprises a light-chain variable region (VL). In some aspects, the antigen-binding region that specifically binds B7-H3 comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). In some embodiments, the heavy-chain variable region comprises three complementarity-determining regions (CDRs) from the heavy-chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light-chain variable region comprises three complementarity-determining regions (CDRs) from the light-chain variable region, LCDR1, LCDR2, and LCDR3.

[0283] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, and / or VL comprised by the antigen-binding region that specifically binds B7-H3 of the present invention are respectively defined as herein.

[0284] In some embodiments, the heavy-chain variable region of the antigen-binding region that specifically binds B7-H3 of the present invention

[0285] (i) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:4 or consists of said amino acid sequence; or

[0286] (ii) comprises the amino acid sequence of SEQ ID NO:4 or consists of said amino acid sequence; or

[0287] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4 or consists of said amino acid sequence, and preferably, said amino acid alterations do not occur in the CDR regions.

[0288] In some embodiments, the light chain variable region of the antigen-binding region that specifically binds to B7-H3 according to the present invention

[0289] (i) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10 or consists of said amino acid sequence; or

[0290] (ii) comprises the amino acid sequence of SEQ ID NO: 10 or consists of said amino acid sequence; or

[0291] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10 or consists of said amino acid sequence, and preferably, said amino acid alterations do not occur in the CDR regions.

[0292] In some embodiments, the three complementarity-determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to B7-H3 according to the present invention, HCDR1, HCDR2, and HCDR3, are selected from

[0293] (i) the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 4, or

[0294] (ii) a sequence that, relative to the sequence in (i), contains a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in said three HCDR regions,

[0295] wherein the HCDRs can be determined according to any CDR-determining scheme, for example, determined according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0296] for example, said HCDR1 is determined by the Abm scheme, and said HCDR2 and HCDR3 are determined by the Kabat scheme.

[0297] In some embodiments, the three complementarity-determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to B7-H3 according to the present invention, LCDR1, LCDR2, and LCDR3, are selected from

[0298] (i) the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 10, or

[0299] (ii) Sequences that, relative to the sequences in (i), contain a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions

[0300] wherein the LCDR can be determined respectively according to any CDR-determining scheme, for example, determined respectively according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0301] For example, the LCDR1-3 are determined respectively by the Kabat scheme.

[0302] In some embodiments, the antigen-binding region that specifically binds to B7-H3 of the present invention comprises three complementarity-determining regions (HCDR) contained in the heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 and three complementarity-determining regions (LCDR) contained in the light-chain variable region consisting of the amino acid sequence of SEQ ID NO: 10;

[0303] wherein the HCDR and LCDR can be determined respectively according to any CDR-determining scheme, for example, determined respectively according to the Kabat, AbM, Chothia, Contact, or IMGT scheme or a combination thereof;

[0304] For example, the HCDR1 adopts the Abm scheme, and the HCDR2, HCDR3, and LCDR1-3 are determined respectively by the Kabat scheme.

[0305] In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence, or the HCDR1 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 1.

[0306] In some embodiments, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence, or the HCDR2 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 2.

[0307] In some embodiments, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, or consists of the amino acid sequence, or the HCDR3 comprises an amino acid sequence having one, two, or three changes (preferably an amino acid substitution, preferably a conservative substitution) compared with the amino acid sequence of SEQ ID NO: 3.

[0308] In some embodiments, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or the LCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

[0309] In some embodiments, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:8, or the LCDR2 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0310] In some embodiments, the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:9, or the LCDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

[0311] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0312] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0313] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein

[0314] HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:2, HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:3, LCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:7, LCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8, and LCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:9.

[0315] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises

[0316] HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3; LCDR1 as shown in SEQ ID NO:7, LCDR2 as shown in SEQ ID NO:8, and LCDR3 as shown in SEQ ID NO:9.

[0317] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:10 or consists of the amino acid sequence.

[0318] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention comprises VH and VL, wherein VH and VL respectively comprise the amino acid sequences shown below or consist of the amino acid sequences: SEQ ID NO:4 and SEQ ID NO:10.

[0319] In some specific embodiments of the present invention, the antigen-binding region specifically binding to B7-H3 of the present invention is a Fab.

[0320] II-3 Fab fragment

[0321] In some embodiments, the first antigen-binding region and / or the second antigen-binding region of the present invention is a Fab fragment. The Fab fragment suitable for use as an antigen-binding region of a multispecific antibody such as a bispecific antibody described herein consists of two polypeptide chains comprising the antibody VH, CH1, VL, and CL domains, wherein the VH pairs with the VL and the CH1 pairs with the CL to form an antigen-binding region.

[0322] In some embodiments, in a Fab, one chain contains VH and CH1 from the N-terminus to the C-terminus (i.e., VH-CH1) or consists of VH and CH1, and the other chain contains VL and CL from the N-terminus to the C-terminus (i.e., VL-CL) or consists of VL and CL. In some embodiments, in the multispecific or bispecific antibodies of the present invention, the Fab can be connected to the N-terminus of the Fc domain of the antibody through the C-terminus of the chain containing VH. Preferably, the Fab contains a VH-CH1 chain and a VL-CL chain and can be connected to the antibody Fc domain through the C-terminus of CH1 of the VH-CH1 chain. In some embodiments, the connection is a direct connection or through a linker. In this context, the Fab chain containing VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain containing VL-CL is also referred to as the Fab light chain.

[0323] In some embodiments, the CH1 is CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1

[0324] (i) contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 32 or consists of the amino acid sequence;

[0325] (ii) contains the amino acid sequence of SEQ ID NO: 32 or consists of the amino acid sequence; or

[0326] (iii) contains an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 32 or consists of the amino acid sequence.

[0327] In some embodiments, the CL is the kappa light chain constant region or the lambda light chain constant region. In some embodiments, the CL is the kappa light chain constant region. In some embodiments, the CL

[0328] (i) contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 11 or consists of the amino acid sequence;

[0329] (ii) contains the amino acid sequence of SEQ ID NO: 11 or consists of the amino acid sequence; or

[0330] (iii) Comprising an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:11, or consisting of said amino acid sequence. In some embodiments, the first antigen-binding region is a Fab that specifically binds to TROP2, wherein the Fab fragment is from an anti-TROP2 antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-TROP2 antibody. In some embodiments, the Fab that specifically binds to TROP2 as the first antigen-binding region comprises the 6 CDRs of the antigen-binding region that specifically binds to TROP2 described herein. In some embodiments, the Fab that specifically binds to TROP2 as the first antigen-binding region comprises the VH or VL of the antigen-binding region that specifically binds to TROP2 described herein, or comprises the VH and VL of the antigen-binding region that specifically binds to TROP2 described herein.

[0331] In some embodiments, the second antigen-binding region is a Fab that specifically binds to B7-H3, wherein the Fab fragment is from an anti-B7-H3 antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-B7-H3 antibody. In some embodiments, the Fab that specifically binds to B7-H3 as the second antigen-binding region comprises the 6 CDRs of the antigen-binding region that specifically binds to B7-H3 described herein. In some embodiments, the Fab that specifically binds to B7-H3 as the second antigen-binding region comprises the VH or VL of the antigen-binding region that specifically binds to B7-H3 described herein, or comprises the VH and VL of the antigen-binding region that specifically binds to B7-H3 described herein.

[0332] II-4 Fc region

[0333] In some embodiments, the bispecific antibody of the present invention further comprises an Fc region, wherein the included Fc regions may be the same or different.

[0334] In some embodiments, the antibody molecule of the present invention comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0335] In some embodiments, the first Fc region and the second Fc region are different and can dimerize to form a heterodimeric Fc scaffold.

[0336] In this text, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include a native sequence Fc region and a variant Fc region. The native sequence F region encompasses various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present invention comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region may also carry an IgG hinge region or a partial IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a partial IgG1 hinge region, such as the sequence from D221 to P230 according to the EU numbering. Mutations may be contained in the hinge region.

[0337] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0338] In some embodiments, the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises the amino acid sequence SEQ ID NO:30 or SEQ ID NO:31 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 98%, 99% or higher identity, to the amino acid sequence or consisting of the amino acid sequence.

[0339] As understood by those skilled in the art, in order to facilitate the formation of the multispecific antibody of the present invention as a heterodimer, the Fc regions contained in the multispecific antibody of the present invention may contain mutations that facilitate the heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0340] Methods for promoting Fc region heterodimerization are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementarily engineered CH3 regions (such that the CH3 regions of the first and second Fc regions heterodimerize and no homodimers are formed between two first CH3 regions or two second CH3 regions).

[0341] Preferably, based on the Knob-into-hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. See, for example, 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). In some embodiments, the Knob mutation and the Hole mutation may further comprise a cysteine mutation and thus introduce a disulfide bond. The disulfide bond can be used to stabilize the final antibody product or the ADC of the antibody.

[0342] In a particular embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (Knob mutation); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (Hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to the EU index).

[0343] In some embodiments, the Fc region may also contain cysteine residue substitutions respectively, so as to obtain non-natural disulfide bond linkages. In some embodiments, the serine residue at position 354 in one Fc region is replaced with a cysteine residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue), while in another Fc region, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering is according to the EU index). Therefore, in yet another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering is according to the EU index), and optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering is according to the EU index).

[0344] In a specific embodiment, one Fc region contains the amino acid substitution T366W, and another Fc region contains the amino acid substitutions T366S, L368A and Y407V (numbering is according to the EU index).

[0345] In a specific embodiment, one Fc region contains the amino acid substitutions S354C and T366W, and another Fc region contains the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering is according to the EU index).

[0346] Corresponding mutations can also be introduced into the first Fc region and the second Fc region based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141, which is incorporated herein by reference in its entirety.

[0347] In a particular embodiment,

[0348] The first CH3 region contains the S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region contains the S364R / K mutation and the second CH3 region contains the K370S / T / A / V mutation. In some embodiments, the first CH3 region contains the S364R mutation and the second CH3 region contains the K370S mutation.

[0349] In some embodiments, the first CH3 region contains the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region contains S364R / K + D399K / R, and the second CH3 region contains K370S / T / A / V + Y349T / S / A / V. In some embodiments, the first CH3 region contains S364R + D399K, and the second CH3 region contains K370S + Y349T. In some embodiments, the first CH3 region further contains E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further contains K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0350] In some embodiments, the first CH3 region contains S364R + D399K, and the second CH3 region contains K370S + Y349T + K409D. In some embodiments, the first CH3 region further contains E357N. In some embodiments, the second CH3 region further contains Q347D. In some embodiments, the first CH3 region further contains E357N, and the second CH3 region further contains Q347D. In some embodiments, the first CH3 region and the second CH3 region further contain T350V respectively, or both contain T350V.

[0351] Thus, in some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K + E357N, and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K + E357N + T350V, and the second CH3 region comprises K370S + Y349T + K409D + Q347D + T350V.

[0352] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D + T411R / K, and the second CH3 region comprises D399K / R + K370T / S / A / V. In some embodiments, the CH3 region comprises K409E + T411R, and the second CH3 region comprises D399K + K370T.

[0353] In some specific embodiments, the first and second CH3 regions have the following mutation combinations:

[0354] First CH3 region Second CH3 region S364R, D399K, E357N, T350V K370S, Y349T, Q347D, K409D, T350V K409E, T411R D399K, K370T S364R, D399K, E357N K370S, Y349T, Q347D, K409D S364R, D399K K370S, Y349T, Q347D, K409D S364R, D399K K370S, Y349T, K409D

[0355] The Fc region in the antibodies of the present invention can also be mutated to obtain desired properties. Mutations of the Fc region are known in the art.

[0356] In one embodiment, the Fc region is modified in terms of the properties of the effector functions of the Fc region (such as the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from: using an Fc isotype or Fc region modification that naturally has a reduced or eliminated effector function.

[0357] In a preferred embodiment, the Fc region has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions, e.g., comprising mutations that achieve the above functions.

[0358] As understood by those skilled in the art, depending on the intended use of the antibody molecules of the present invention, the antibody molecules of the present invention may also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation. In yet another preferred embodiment, the Fc fragment may have mutations that result in an increased serum half-life, such as mutations that improve the binding of the Fc fragment to FcRn. In some embodiments, the Fc fragment comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:29 and comprising the L234A / L235A mutation.

[0359] Thus, in a specific embodiment, the two Fc regions comprised in a multispecific antibody of the present invention, such as a bispecific antibody, are heterodimerized, wherein one Fc-region polypeptide comprises the mutations S354C and T366W, and the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V.

[0360] Thus, in a specific embodiment, the two Fc regions comprised in a multispecific antibody of the present invention, such as a bispecific antibody, are heterodimerized, wherein one Fc-region polypeptide comprises the amino acid sequence shown in SEQ ID NO:27 or consists thereof, and the other Fc-region polypeptide comprises the amino acid sequence shown in SEQ ID NO:28 or consists thereof.

[0361] Thus, in a specific embodiment, the two Fc regions comprised in a multispecific antibody of the present invention, such as a bispecific antibody, are heterodimerized, wherein one Fc-region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:27, and the other Fc-region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:28.

[0362] Thus, in a specific embodiment, the two Fc regions of the multispecific antibody of the present invention, such as a bispecific antibody, are heterodimerized. One Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 27 and comprises the mutations S354C and T366W, and optionally the L234A / L235A mutation, while the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 28 and comprises the mutations Y349C, T366S, L368A and Y407V, and optionally the L234A / L235A mutation.

[0363] In some embodiments, the Fc region further comprises other mutations that facilitate the purification of the heterodimer.

[0364] II-5 Exemplary bispecific antibody molecule

[0365] In some embodiments, the anti-B7-H3 / TROP2 bispecific antibody of the present invention has one or more of the following properties:

[0366] (1) The bispecific antibody of the present invention can specifically bind to cells expressing TROP2 and / or B7-H3, such as tumor cells;

[0367] (2) The bispecific antibody of the present invention can be internalized by cells, such as tumor cells, and for example, its internalization activity is stronger than that of the parental monoclonal antibody.

[0368] In some embodiments, in the antibody molecule of the present invention, the heavy chain variable region of the antigen-binding region that specifically binds TROP2 as described herein is linked to the heavy chain constant region CH, for example, the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecule of the present invention, the light chain variable region of the antigen-binding region that specifically binds TROP2 as described herein is linked to the light chain constant region, for example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecule of the present invention, in the antigen-binding region that specifically binds TROP2 as described herein, the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL.

[0369] In some embodiments, in the antibody molecules of the present invention, the heavy chain variable region of the antigen-binding region that specifically binds B7-H3 as described herein is linked to the heavy chain constant region CH, for example, the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecules of the present invention, the light chain variable region of the antigen-binding region that specifically binds B7-H3 as described herein is linked to the light chain constant region CL, for example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecules of the present invention, in the antigen-binding region that specifically binds B7-H3 as described herein, the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL.

[0370] In some embodiments, the heavy chain constant region comprises CH1 and the Fc region as described herein, linked via or without a hinge region. In some embodiments, the heavy chain constant region consists of the CH1 and the Fc region.

[0371] In some preferred embodiments, the present invention provides a bispecific antibody molecule comprising a Fab fragment that specifically binds TROP2 as described herein, a Fab fragment that specifically binds B7-H3 as described herein, and an Fc dimer as described herein, wherein the Fab fragment that specifically binds TROP2 forms a semi-antibody that specifically binds TROP2 with an Fc as described herein, and the Fab fragment that specifically binds B7-H3 forms a semi-antibody that specifically binds B7-H3 with an Fc as described herein. In some embodiments, the bispecific antibody is an IgG-like antibody having the configuration as Figure 1 shown. In a specific embodiment, the first Fc region and the second Fc region comprise a Knob-into-Hole mutation, and optionally a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation.

[0372] In some embodiments, the bispecific antibody comprises or consists of heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein heavy chain 1 and light chain 1 constitute the first semi-antibody, and heavy chain 2 and light chain 2 constitute the second semi-antibody; wherein heavy chain 1 comprises the heavy chain variable region of the first antigen-binding region and the first heavy chain constant region; light chain 1 comprises the light chain variable region of the first antigen-binding region and the first light chain constant region; and heavy chain 2 comprises the heavy chain variable region of the second antigen-binding region and the second heavy chain constant region; light chain 2 comprises the light chain variable region of the second antigen-binding region and the second light chain constant region.

[0373] In a specific embodiment, the bispecific antibody of the present invention specifically binds TROP2 and B7-H3 and comprises or consists of the following:

[0374] Heavy chain 1: From the N-terminus to the C-terminus, it comprises or consists of: a Fab heavy chain variable region that specifically binds to TROP2 - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected to the N-terminus of the first Fc region at its C-terminus via or without a linker (such as a hinge region);

[0375] Light chain 1: From the N-terminus to the C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to TROP2 - a light chain constant region;

[0376] Heavy chain 2: From the N-terminus to the C-terminus, it comprises or consists of: a heavy chain variable region of a Fab that specifically binds to B7-H3 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected to the N-terminus of the second Fc region at its C-terminus via or without a linker (such as a hinge region);

[0377] Light chain 2: From the N-terminus to the C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to B7-H3 - a light chain constant region,

[0378] Preferably, each domain is directly connected;

[0379] Optionally, the first Fc region contains the mutations S354C and T366W, and the second Fc region contains Y349C, T366S, L368A, and Y407V or vice versa; optionally, the first Fc region and the second Fc region further respectively contain mutations that reduce binding to Fcγ receptors, such as the L234A / L235A mutation.

[0380] The domains of the bispecific antibody of the present invention are described herein, such as Fab, Fc, VH, and VL as described herein.

[0381] In some specific embodiments, the bispecific antibody of the present invention that specifically binds to TROP2 and B7-H3 comprises or consists of:

[0382] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence; and light chain 2 comprises the amino acid sequence shown in SEQ ID NO:12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto or consisting of said amino acid sequence.

[0383] Preparation method of II-6

[0384] In some embodiments, the present invention provides a method for preparing an antibody molecule of the present invention, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody molecule of the present invention, and optionally isolating the antibody. In a certain embodiment, the method further comprises recovering the antibody molecule of the present invention from the host cell.

[0385] In some embodiments, a method for preparing an antibody molecule of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for the expression of the antibody or its chains, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0386] In some embodiments, the method for producing a bispecific antibody of the present invention comprises the steps of: culturing a host cell comprising nucleic acids encoding the respective chains of the molecule under conditions suitable for expressing the respective chains of the molecule, to produce the bispecific antibody of the present invention.

[0387] For the recombinant production of an antibody molecule of the present invention, a nucleic acid encoding the antibody (e.g., the antibody described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0388] In some embodiments, the nucleic acids of the invention encode any one of the chains of the antibody molecules of the invention. The invention relates to nucleic acids encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptides encoded by said nucleic acids are capable of displaying human TROP2 / or B7-H3 antigen-binding ability.

[0389] As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the invention can be generated using methods well known in the art, such as by de novo solid-phase DNA synthesis or by PCR amplification.

[0390] In some embodiments, the nucleic acids encoding the individual chains of the bispecific antibody can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding the individual chains of the bispecific antibody can be introduced into the same or different host cells for expression.

[0391] In some embodiments, the invention also relates to vectors comprising the nucleic acids of the invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0392] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are cultured in a medium and screened for suitable activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and can be varied or optimized based on the methods known from this specification and the prior art, depending on the particular expression vector and mammalian host cell used.

[0393] Additionally, cells that have stably incorporated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of the transfected host cells.

[0394] In some embodiments, the host cell comprises the nucleic acid of the present invention or comprises the expression vector of the present invention. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells or Expi293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as a bacterium, such as Escherichia coli.

[0395] The antibody molecules prepared as described herein can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0396] III. Antibody-drug conjugates

[0397] The present invention provides an antibody-drug conjugate having the formula (I):

[0398] Ab-(L-D) p (I)

[0399] or a pharmaceutically acceptable salt or solvate thereof,

[0400] wherein:

[0401] Ab is an antibody or a fragment thereof (e.g., an antigen-binding fragment) that specifically binds to TROP2 and B7-H3 (e.g., human TROP2 and human B7-H3);

[0402] L is a linker;

[0403] D is a drug, preferably an anti-tumor compound; and

[0404] p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0405] It should be understood that p means the number of -L-P groups to which Ab is attached in the antibody-drug conjugate molecule of formula (I).

[0406] In some embodiments, Ab in formula (I) of the present invention is the antibody of the present invention, such as a bispecific antibody.

[0407] In some embodiments, D in formula (I) of the present invention can be any anti-tumor compound, as long as it is a compound with anti-tumor effects and having substituents or partial structures capable of being linked to the linker structure, without particular limitation. For example, the anti-tumor compound can be a pharmaceutically active compound having an effect on tumors. For anti-tumor compounds, preferably part or all of the linker can be cleaved within tumor cells, releasing the anti-tumor compound moiety, thereby exhibiting anti-tumor effects. When cleaving the linker at the connection part with the drug, the anti-tumor compound is released in an unmodified or modified structure, and its anti-tumor effects can be exerted.

[0408] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent or a chemotherapeutic agent, such as camptothecin compounds like exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE), maytansine compounds such as the small molecule microtubule inhibitor DM1, taxanes such as paclitaxel or docetaxel, anthracyclines, epothilones, mitomycin, combretastatin, vinca alkaloids, calicheamicin, doxamicin, microtubulysin, phalloidin, bleomycin, MEK inhibitors, KSP inhibitors, etc.

[0409] In some embodiments, D is as shown in formula (D-1):

[0410]

[0411] wherein R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl, and C2-C6 haloalkynyl;

[0412] R 2 is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and -SR 4 ; R 3 is selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, and -OR 4 ; or R 2 and R 3 together form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0413] R 4 is selected from H or C1-C4 alkyl.

[0414] In some embodiments, R 1is H, R 2 is a C1-C6 alkyl group, R 3 is -F.

[0415] In some embodiments, D is as shown in formula (D-2):

[0416]

[0417] wherein R 1 , R 2 and R 3 are as defined above.

[0418] In some embodiments, D is as shown in formula (D-3):

[0419]

[0420] In some embodiments, D is as shown in formula (D-4):

[0421]

[0422] In some embodiments, -L- is the following structure: -Z-E-NH-CH2-Q-L 2 -L 1 -

[0423] where Z is connected to Ab, and L 1 is connected to D;

[0424] Z is selected from where m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8, preferably 5; the carbonyl group at its right end is covalently connected to E;

[0425] E is a peptide containing 2-10 amino acids, wherein the peptide is optionally substituted by one or more polyol groups, and the N-terminus of the peptide is covalently connected to Z;

[0426] Q is -O- or -S-;

[0427] L 1 is absent or -(C1-C 10 alkylene)-;

[0428] L 2 is absent, -N(R 5 )C(O)-(C1-C 10 alkylene)-* or -C(O)N(R 5 )-(C1-C 10 alkylene)-*; where * indicates that the terminus is covalently connected to Q; and

[0429] R 5is H or a C1-C6 alkyl group.

[0430] In some embodiments, E is a peptide residue composed of 2, 3, or 4 amino acids.

[0431] In some embodiments, the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group.

[0432] In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala- or wherein R 6 is H or a C1-C6 alkyl group, and wherein these E groups are covalently linked to Z through the left N-terminus.

[0433] In some embodiments, -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10 alkylene)-*, where * indicates that the terminus is covalently linked to Q;

[0434] R 5 is H or a C1-C6 alkyl group.

[0435] In some embodiments, -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0436] In some embodiments, -Z-E-NH-CH2-Q-L 2 -L 1 - has the following structure

[0437]

[0438] It should be understood that the right end of this structure is connected to the D part.

[0439] In some embodiments, the payload (e.g., drug) of the immunoconjugate of the present invention is attached to the modified antibody or antibody fragment through the thiol group of a free cysteine of the antibody or its antigen-binding fragment (optionally via a linker). In some embodiments, the payload (e.g., drug) of the immunoconjugate of the present invention is linked to the thiol group of the cysteine through a cleavable or non-cleavable linker.

[0440] In some embodiments, Ab is linked to L through the sulfur atom on its thiol group, and the antibody-drug conjugate of formula (I) can be represented by the following formula:

[0441] Ab’-(S-L-D) p (I’)

[0442] wherein Ab’ is as defined above for Ab, and L, D, and p are as defined above.

[0443] In some embodiments, the antibody-drug conjugate has an average DAR value of 5-11 or 7.5-8.5.

[0444] In some embodiments, the antibody-drug conjugate is selected from

[0445]

[0446] wherein Ab is the bispecific antibody of the present invention, preferably HZ5C2.9 / hRS7.13; q is as defined above for p,

[0447] Preferably, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR of, for example, 5-11, 6-10, 7-9, or 7.5-8.5.

[0448] It should be understood that the S atom linked to Ab in the above ADC is derived from the antibody Ab. Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a thiol group -SH, which then links to the maleimide moiety of the linker.

[0449] IV. Preparation method of the ADC of the present invention

[0450] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. The "ADC" in the present invention is defined as an antibody conjugated to a biologically and / or pharmaceutically active substance (D) through a linker (L). The method includes conjugating the antibody (Ab) of the present invention to one or more active substances D through one or more linkers (L) defined in the present invention.

[0451] In some embodiments, the method includes preparing Ab for the ADC, which includes culturing a host cell containing a nucleic acid encoding the Ab (such as any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid under conditions suitable for the expression of Ab or its chain, as provided above, and optionally recovering the Ab from the host cell (or the host cell culture medium).

[0452] In some embodiments, the method includes the following steps:

[0453] (a) Add antibody Ab to a buffer solution, add a reducing agent, and then incubate;

[0454] (b) Add a linker-payload to the reaction solution in step (a) for conjugation to obtain a crude product; and

[0455] (c) Optionally purify the crude product to obtain the antibody-drug conjugate of the present invention;

[0456] wherein Ab is as defined above.

[0457] It should be understood that the linker-payload reacts with Ab to provide the -L-D moiety in the compound of formula I, and when -L-D is clearly defined, the structure of the linker-payload can be determined or selected.

[0458] In some embodiments, the buffer solution in step a) is a PBS buffer, preferably having a pH of 5.0 - 9.0, such as 6.0 - 8.0.

[0459] In some embodiments, the reducing agent in step a) is TCEP.

[0460] In some embodiments, the linker-payload has the following structure: Z’-E-NH-CH2-Q-L 2 -L 1 -D, where E, Q, L 2 、L 1 、D are as defined above, and Z’ is m is as defined above.

[0461] In some embodiments, the steps are carried out under the specific reaction conditions disclosed in the examples.

[0462] It should be noted that embodiments obtained by floating the range or specific values of the specific reaction conditions disclosed in the examples by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated in the present invention.

[0463] In some embodiments, the ADC can be prepared according to the schematic diagram as Figure 8 shown. The ADCs obtained are also contemplated in the present invention.

[0464] V. Pharmaceutical Composition

[0465] In some embodiments, the present invention provides a composition comprising any of the bispecific antibody molecules and / or ADC molecules described herein or a pharmaceutically acceptable salt thereof, preferably the composition is a pharmaceutical composition or a pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., the pharmaceutical composition, comprises a bispecific antibody molecule and / or an ADC molecule of the present invention, and a combination of one or more other therapeutic agents.

[0466] The present invention also includes a composition (including a pharmaceutical composition) comprising a salt of a bispecific antibody molecule or a fragment thereof of the present invention. The present invention also includes a composition (including a pharmaceutical composition) comprising an ADC molecule of the present invention or a pharmaceutically acceptable salt thereof. These compositions may also comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients known in the art, including buffers.

[0467] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, etc. that are physiologically compatible.

[0468] For the use and application of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", 8th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0469] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposome formulations and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0470] A drug comprising a bispecific antibody molecule or an ADC as described herein can be prepared by mixing a bispecific antibody molecule or an ADC of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0471] The pharmaceutical composition or formulation of the present invention may also comprise more than one active ingredient, which is required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0472] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing an antibody or ADC molecule, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0473] VI. Drug Combinations and Kits

[0474] In some embodiments, the present invention also provides a drug combination or drug combination product, which comprises a bispecific antibody molecule and / or an ADC molecule of the present invention, and one or more other therapeutic agents (such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), etc.).

[0475] Another object of the present invention is to provide a kit, which comprises the drug combination of the present invention, preferably the kit is in the form of a drug dosage unit. Thus, dosage units can be provided according to the dosing regimen or drug administration interval.

[0476] In one embodiment, the kit of the present invention comprises in the same package:

[0477] - a first container containing a pharmaceutical composition comprising a bispecific antibody molecule and / or an ADC molecule of the present invention;

[0478] - a second container containing a pharmaceutical composition comprising other therapeutic agents.

[0479] VII. Uses and Methods

[0480] On the one hand, the present invention provides a method for preventing or treating a disease in a subject, which comprises administering to the subject an effective amount of a bispecific antibody molecule, ADC molecule, pharmaceutical composition, drug combination or kit of the present invention.

[0481] In some embodiments, the disease is a B7-H3 and / or TROP2-related disease. In some embodiments, the disease is related to the abnormal expression or abnormal activity of B7-H3 and / or TROP2.

[0482] In some embodiments, the disease is a tumor, such as cancer.

[0483] In some embodiments, the tumor, such as cancer, is B7-H3 positive, e.g., it comprises tumor cells that express B7-H3. In some embodiments, the patient's tumor comprises tumor cells that express B7-H3. In some embodiments, the patient's tumor cells express B7-H3, e.g., moderately express B7-H3, preferably highly express B7-H3. In some embodiments, the tumor (such as cancer) patient's tumor tissue has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) B7-H3, e.g., compared to the B7-H3 level in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) B7-H3, e.g., compared to the B7-H3 level in the same cells of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0484] In some embodiments, the tumor, such as cancer, is TROP2 positive, e.g., it comprises tumor cells that express TROP2. In some embodiments, the patient's tumor comprises tumor cells that express TROP2. In some embodiments, the patient's tumor cells express TROP2, e.g., moderately express TROP2, preferably highly express TROP2. In some embodiments, the tumor (such as cancer) patient's tumor tissue has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) TROP2, e.g., compared to the TROP2 level in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels of, e.g., nucleic acid or protein levels or activity of) TROP2, e.g., compared to the TROP2 level in the same cells of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0485] In some embodiments, the tumor, such as cancer, is B7-H3 positive and TROP2 positive, e.g., it comprises tumor cells that express B7-H3 and TROP2. In some embodiments, the patient's tumor comprises tumor cells that express B7-H3 and TROP2. In some embodiments, the patient's tumor cells express B7-H3 and TROP2, e.g., moderately or highly express B7-H3 and TROP2. In some embodiments, the tumor (such as cancer) in the patient's tumor tissue has (e.g., elevated levels of, e.g., nucleic acid or protein levels or activities of) B7-H3 and TROP2, e.g., compared to the levels of B7-H3 and TROP2 in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels of, e.g., nucleic acid or protein levels or activities of) B7-H3 and TROP2, e.g., compared to the levels of B7-H3 and TROP2 in the same cells of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0486] In some embodiments, the tumor, such as cancer, includes solid tumors, hematological tumors, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, middle, or late stage or be metastatic cancer.

[0487] In a specific embodiment, the ADC molecule of the present invention can kill tumor cells and / or inhibit tumor cell proliferation, and the tumor cells are, for example, tumor cells that express B7-H3 and / or TROP2, such as lung cancer cells including non-small cell lung cancer and small cell lung cancer cells, colon cancer cells, oral squamous cell carcinoma cells, breast cancer cells, melanoma cells, head and neck tumor cells, prostate cancer cells, esophageal cancer cells, cervical cancer cells, renal cancer cells, bladder cancer cells, ovarian cancer cells, or pancreatic cancer cells.

[0488] In some embodiments, the tumor is tumor immune escape.

[0489] In some embodiments, the tumor is cancer, such as lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumor, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer, or pancreatic cancer.

[0490] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or is at risk of suffering from a disease described herein. In some embodiments, the subject has received or has been receiving other treatments, such as chemotherapy treatment and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0491] In other aspects, the present invention provides the use of the above-mentioned bispecific antibody molecule, ADC molecule or pharmaceutical composition or drug combination or kit in the production or preparation of a drug for the uses described herein, e.g., for preventing or treating related diseases or disorders mentioned herein.

[0492] In other aspects, the present invention provides the above-mentioned bispecific antibody molecule, ADC molecule or pharmaceutical composition or drug combination or kit for use in therapy, e.g., for the uses described herein, e.g., for preventing or treating related diseases or disorders mentioned herein.

[0493] In some embodiments, the bispecific antibody molecule, ADC molecule or pharmaceutical composition or drug combination or kit of the present invention delays the onset of a disorder and / or symptoms associated with the disorder.

[0494] In some embodiments, the bispecific antibody molecule, ADC molecule or pharmaceutical composition of the present invention can also be administered in combination with one or more other therapies such as treatment modalities and / or other therapeutic agents for the uses described herein, e.g., for preventing and / or treating related diseases or disorders mentioned herein.

[0495] In some embodiments, the treatment modality includes surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0496] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0497] Exemplary other antibodies include antibodies that specifically bind to immune checkpoints.

[0498] The combination therapies of the present invention encompass co-administration (e.g., two or more therapeutic agents are included in the same formulation or separate formulations), and separate administration, in which case, the administration of the ADC molecule of the present invention can occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or medicaments.

[0499] The administration route of the pharmaceutical composition is according to known methods, for example, oral, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; through sustained release systems or through implantable devices. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by an implantable device.

[0500] The composition can also be administered locally via an implantable membrane, sponge or another suitable material that absorbs or encapsulates the desired molecule thereon. In certain embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecule via diffusion, timed-release bolus, or continuous administration.

[0501] These and other aspects and embodiments of the invention are described in the accompanying drawings and the following detailed description of the invention and are illustrated in the following examples. Any or all of the features discussed above and throughout this application can be combined in the various embodiments of the invention. The following examples further illustrate the invention, however, it should be understood that the examples are described in an illustrative rather than limiting manner and that various modifications can be made by those skilled in the art. Examples

[0502] Example 1: Expression and purification of recombinant humanized anti-Trop2 / B7-H3 bispecific antibody

[0503] The antibody of the present invention can be expressed and purified substantially as follows. A suitable host cell HEK 293 (which can also be other host cells such as CHO cells) can be transiently or stably transfected with a secretory antibody expression system using an optimal predetermined heavy chain: light chain vector ratio or a single vector system that encodes both the heavy chain and the light chain.

[0504] Specifically, one or more DNA molecules encoding the following amino acid sequences can be utilized: a first heavy chain having the amino acid sequence SEQ ID NO: 6, a first light chain having SEQ ID NO: 12, a second heavy chain having the amino acid sequence SEQ ID NO: 18, and a second light chain having the amino acid sequence SEQ ID NO: 23, and transiently or stably transfected using an expression system secreting antibodies to prepare the bispecific antibody HZ5C2.9 / hRS7.13 of the present invention. Among them, the first heavy chain having the amino acid sequence SEQ ID NO: 6 and the first light chain having SEQ ID NO: 12 together form a first half-antibody, and this first half-antibody specifically binds to B7-H3 or its extracellular domain; and, the second heavy chain having the amino acid sequence SEQ ID NO: 18 and the second light chain having the amino acid sequence SEQ ID NO: 23 together form a second half-antibody, and this second half-antibody specifically binds to Trop2 or its extracellular domain; and, the first half-antibody and the second half-antibody together form the complete bispecific antibody HZ5C2.9 / hRS7.13 of the present invention.

[0505] Figure 1 Figure 4 shows a schematic diagram of the structure of the above antibody.

[0506] One of a variety of common techniques can be used to purify the antibody. For example, the culture medium can be conveniently applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) that has been equilibrated with a compatible buffer such as phosphate buffer (pH 7.4). The column is washed to remove non-specific binding components. The bound antibody can be eluted, for example, by a pH gradient (e.g., 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or phosphate-buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0). The antibody fractions are detected, for example, by ultraviolet absorption or SDS-PAGE, and then the antibody fractions are combined. Further purification is optionally performed according to the intended use. The purified antibody can be concentrated and / or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by conventional techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography, etc. The purified antibody can be immediately frozen below -30°C or lyophilized.

[0507] Expression and purification of specific bispecific antibodies

[0508] Insert the heavy chain sequence and light chain sequence of TROP2-Arm, and the heavy chain sequence and light chain sequence of B7-H3-Arm into the vector pcDNA3.1 (Invitrogen, V790-20) to obtain the heavy chain plasmid and light chain plasmid against the TROP2 end, and the heavy chain plasmid and light chain plasmid against the B7-H3 end, respectively.

[0509] Transiently transfect the heavy chain plasmid and light chain plasmid of the TROP2 end and the heavy chain plasmid and light chain plasmid of the B7-H3 end into ExpiCHO (Invitrogen, A29133) cells, respectively. Harvest the cell fermentation broth after 7 days, filter and clarify it, and capture it with a Hitrap Mabselect Sure chromatography column (GE Healthcare, 11-0034-95) to obtain the TROP2 antibody parent and the B7-H3 antibody parent.

[0510] After detecting the concentration by the A280 method, mix the antibody parents in a molar ratio of 1:1, add an appropriate amount of reducing agent GSH, react overnight at room temperature, ultrafilter to remove the reducing agent, and terminate the reaction. Perform fine purification using a MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01). The anti-B7-H3 parent solution is 20 mM sodium phosphate buffer (pH 6.6), and the anti-TROP2 parent solution is 20 mM sodium phosphate buffer containing 1 M sodium chloride (pH 6.6). The elution gradient is 0-50% (30 column volumes). Ultrafilter and change the buffer of the eluted protein solution to PBS (Gibco, 70011-044), and detect the purity by SEC-HPLC.

[0511] Expression and purification of monospecific antibodies

[0512] First, construct an expression vector. Place the heavy chain variable region and light chain variable region of the anti-B7-H3 humanized antibody HZ5C2.9, the anti-TROP2 antibody hRS7.13, and the control antibody (negative control antibody IgG) (see sequence list information) at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 24) and the light chain kappa constant region (SEQ ID NO: 11), respectively. Then construct them into the pcDNA3.1 expression vector with an N-terminal signal peptide to obtain the heavy and light chain expression vectors. Passage HEK293 cells (Invitrogen) according to the required transfection volume. Adjust the cell density to 1.5×10 6 cells / ml one day before transfection. The cell density on the day of transfection is approximately 3×10 6cells / ml. Take 1 / 10 (v / v) of the final volume of Opti-MEM medium (Gibco catalog number: 31985-070) as the transfection buffer, add the constructed expression vector, mix well, and filter through a 0.22 μm filter head for standby. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid in the previous step (the mass ratio of plasmid to PEI is 1:3), mix well and incubate at room temperature for 10 min to obtain the DNA / PEI mixture. Gently pour the DNA / PEI mixture into HEK293 cells and mix well. After culturing at 37 °C and 8% CO2 for 24 h, supplement with VPA (Sigma, catalog number: P4543-100G) at a final concentration of 2 mM, and 2% (v / v) Feed solution (1 g / L Phytone Peptone + 1 g / L Difco SelectPhytone), and continue culturing for 6 days. After cell culture, centrifuge the cell culture medium at 13,000 rpm for 20 min, collect the supernatant, and purify the supernatant using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95) according to the manufacturer's instructions, and measure the concentration. Take 100 μg of the purified protein, adjust the concentration to 1 mg / mL, and measure the protein purity using a gel filtration chromatography column SW3000 (TOSOH catalog number: 18675).

[0513] Example 2: Determination of the binding kinetics of the bispecific antibody of the present invention to the antigen by the biofilm thin layer interference technique

[0514] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human B7-H3 and human Trop2 was determined by the biofilm thin layer interference measurement technique (ForteBio). The ForteBio affinity measurement was carried out according to the existing method (Estep, P et al., Highthroughput solution Based measurement of antibody antigen affinity andepitope binning. MAbs, 2013.5(2): pp. 270-8).

[0515] Briefly, the sensor was equilibrated offline in the assay buffer for 30 minutes and then the baseline was established by online detection for 60 seconds. The purified antibody obtained as described above was loaded onto the AHQ sensor (ForteBio) for ForteBio affinity measurement online. The sensor with the loaded antibody was then exposed to human B7-H3 protein (Sinobiological, 29849-H08H) or human Trop2 protein (Sinobiological, 90893-C08H), and then the sensor was transferred to the assay buffer for dissociation rate measurement. The K D value was analyzed using ForteBio analysis software.

[0516] Table 1. Affinity constants (equilibrium dissociation constants) of ForteBio-detected antibodies binding to human B7-H3

[0517]

[0518]

[0519] Table 2. Affinity constants (equilibrium dissociation constants) of ForteBio-detected antibodies binding to human Trop2

[0520]

[0521] Example 3: Binding of Trop2 / B7-H3 bispecific antibody to tumor cell lines

[0522] The binding of Trop2 / B7-H3 bispecific antibody HZ5C2.9 / hRS7.13 and parental monoclonal antibodies (HZ5C2.9 and hRS7.13) to human lung cancer cell line NCI-H358 (Cobioer, CBP60136) was determined by flow cytometry (FACS). Specifically, the cells to be detected were counted and diluted to 2×10 6 cells / ml, and 50 μl / well was added to a U-bottom 96-well plate. Centrifuge at 500 g for 5 min to remove the cell culture medium. Trop2 / B7-H3 bispecific antibody HZ5C2.9 / hRS7.13 and parental monoclonal antibodies were added to the U-bottom plate and the cells were resuspended. The starting concentration of 50 μl of antibody per well was 140 nM and then serially diluted 3-fold, with a total of 10 concentration points. Incubate on ice for 30 min. Centrifuge at 500 g for 5 min to remove the supernatant, and wash the cells once with PBS. Add 100 μl of goat anti-human IgG PE-labeled secondary antibody (SouthernBiotech, 51380) to each well. Incubate in the dark on ice for 30 min. Centrifuge at 500 g for 5 min to remove the supernatant, and wash the cells once with PBS. Resuspend the cells with 50 μl of 1×PBS and detect by FACS. The experimental data were analyzed using GraphPad Prism software to obtainFigure 2 。As Figure 2 shown, the binding activity (EC50) of HZ5C2.9 / hRS7.13 is comparable to that of the parental monoclonal antibody, and the total amount of binding (MFI) is higher.

[0523] Example 4: Endocytosis experiment of Trop2 / B7-H3 bispecific antibody

[0524] After digesting NCI-H358 cells, adjust the cell density to 4×10 4 / ml cells / ml; add 50 μl / well to an opaque white 96-well plate and incubate overnight in a 37 °C CO2 incubator; dilute the antibody to be tested to 4 nM with complete medium and use this as the initial concentration for serial dilution at a 1 / 3 ratio; dilute Fab-ZAP (Advanced Targeting Systems, IT-51-250) to 40 nM with complete medium and use this as the initial concentration for serial dilution at a 1 / 3 ratio; take equal volumes of the antibody and Fab-ZAP and mix them 1:1, and incubate at room temperature for 20 min - 30 min; after incubation, take 50 μl / well and add it to the cells, mix well and continue to incubate at 37 °C; on Day 4, take out the plate, add 100 μl of CellTiter-Glo assay reagent to each well, shake at 150 rpm for 2 min on a shaker, and after standing for 10 min, measure the luminescence value using a multifunctional microplate reader. Analyze the experimental data using GraphPad Prism software to obtain Figure 3 。As Figure 3 shown, the endocytosis activity (IC50) of the bispecific antibody HZ5C2.9 / hRS7.13 is stronger than that of the parental monoclonal antibody.

[0525] Example 5: Synthesis of ADC molecule conjugated with NT3

[0526] On the basis of Trop2 / B7-H3 bispecific antibody and the parental monoclonal antibody, an ADC conjugated with a small molecule compound was further designed and synthesized.

[0527] 5.1 Synthesis of compound NT3

[0528]

[0529] According to the method disclosed in Example 4 of WO2021173773A1, compound NT3 was prepared. Its physicochemical data including 1HNMR and mass spectrometry data are consistent with those disclosed in WO2021173773A1.

[0530] 5.2 Synthesis of HZ5C2.9 / hRS7.13-NT3

[0531]

[0532] Where Ab is the antibody HZ5C2.9 / hRS7.13; q is an integer from 4 to 10, such as 8, and the obtained HZ5C2.9 / hRS7.13-NT3 was determined to have an average DAR of 7.98 in the following experiment.

[0533] The specific process is as follows:

[0534] Preparation of HZ5C2.9 / hRS7.13-NT3

[0535] (a) Dissolve the antibody HZ5C2.9 / hRS7.13 prepared according to the method of Example 1 in PBS buffer (Thermofisher, 10010023). Add a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and place the reaction mixture at room temperature for reaction for 2 - 4 hours, where

[0536] (i) The optimal concentration of HZ5C2.9 / hRS7.13 is 2 - 10 mg / mL,

[0537] (ii) The optimal molar ratio of TCEP / Ab is 10.0 - 20.0,

[0538] (iii) The optimal reaction temperature is 20 - 37 °C,

[0539] (iv) The optimal pH value of the reaction is between 6.0 and 8.0.

[0540] (b) Add an excess of linker-toxin (NT3, dissolved in DMSO) to react with the reduced antibody in step (a), and place the reaction mixture at room temperature for 1 - 2 hours, where

[0541] (i) The optimal molar ratio of NT3 / Ab is 10.0 - 16.0,

[0542] (ii) The optimal reaction temperature is 20 - 37 °C to obtain the crude ADC product.

[0543] (c) The obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product HZ5C2.9 / hRS7.13-NT3.

[0544] (d) Using RP-HPLC, calculate the DAR value according to the peak area of each peak at UV280 nm. After calculation, the average DAR of HZ5C2.9 / hRS7.13-NT3 is 7.98.

[0545] (e) Using SEC-HPLC, the purity of the ADC was obtained according to the peak area ratio of the monomer peak, aggregate, and oligomer at UV280 nm. After calculation, the purity of HZ5C2.9 / hRS7.13-NT3 was 98.96%.

[0546] 5.3 Synthesis of HZ5C2.9-NT3

[0547] The preparation process was similar to that of HZ5C2.9 / hRS7.13-NT3, except that the bispecific antibody was replaced with the parental B7-H3 monoclonal antibody HZ5C2.9. Using the same analytical method as HZ5C2.9 / hRS7.13-NT3, the average DAR was measured to be 7.85 and the purity was 97.27%.

[0548] 5.4 Synthesis of hRS7.13-NT3

[0549] The preparation process was similar to that of HZ5C2.9 / hRS7.13-NT3, except that the bispecific antibody was replaced with the parental Trop2 monoclonal antibody hRS7.13. Using the same analytical method as HZ5C2.9 / hRS7.13-NT3, the average DAR was measured to be 7.85 and the purity was 97.14%.

[0550] 5.5 Synthesis of Control IgG1-NT3

[0551] The preparation process was similar to that of HZ5C2.9 / hRS7.13-NT3, except that the bispecific antibody was replaced with the control antibody IgG. Using the same analytical method as HZ5C2.9 / hRS7.13-NT3, the average DAR was measured to be 7.94 and the purity was 99.35%.

[0552] Example 6: Cell Binding Experiment of Trop2 / B7-H3 Bispecific Antibody ADC Molecule

[0553] To detect whether small molecule conjugation would change the binding characteristics of the Trop2 / B7-H3 bispecific antibody to target cells, we used the human lung cancer cell line NCI-H322 (Cobioer, CBP60134) and Trop2&B7-H3 negative cells KM12 (Meisen, CTCC-001-0684) to detect the binding activity of HZ5C2.9 / hRS7.13-NT3 to the target by flow cytometry. The experimental method was the same as that in Example 3.

[0554] The results are as Figure 4It was shown that the binding activities of HZ5C2.9 / hRS7.13-NT3 and HZ5C2.9 / hRS7.13 to NCI-H322 were similar, and neither bound to the non-target cell KM12, indicating that the binding of the antibody was dependent on the expression specificity of the target, and the conjugation of the small molecule NT1 (Payload) did not affect the binding of the antibody.

[0555] Example 7: In vitro cytotoxic effect of Trop2 / B7-H3 bispecific ADC molecules

[0556] Using the Cell Titer-Glo (Promega, G9242) detection kit, the effect of the ADC molecule on cell viability was detected on multiple tumor cell lines co-expressing Trop2&B7-H3: NCI-H358, NCI-H322, SUM149PT (Meisen, CTCC-001-0949), SCC25 (Cobioer, CBP61033), and the normal cell MCF10A (Cobioer, CBP60419).

[0557] After digesting the cells used with Trypsin, adjusting the density, evenly plating them into 96-well plates (Table 3) and adding samples with gradient-diluted concentrations: Trop2 / B7-H3 bispecific ADC molecules and parental monoclonal antibody ADCs, and placing them in an incubator at 37°C for 5 days. After 5 days, 100 μl of Cell Titer Glo detection reagent was added to each well, incubated at room temperature for 30 min, and detected with an enzyme-linked immunosorbent assay reader. Calculate the relative cell viability (relative cell viability = experimental well / control well * 100%), and fit the curve with Graph Pad Prism 8.0.

[0558] As Figure 5 shown, the cytotoxic effects of Trop2 / B7-H3 bispecific ADC molecules on multiple tumor cell lines were stronger than those of the parental monoclonal antibody ADCs. In the normal cell MCF10A, the Trop2 monoclonal antibody ADC hRS7.13-NT3 had strong cytotoxicity, while the cytotoxicity of the bispecific ADC HZ5C2.9 / hRS7.13-NT3 was significantly weaker than that of hRS7.13-NT3; indicating that the bispecific ADC had a better tumor suppression effect than the parental monoclonal antibody ADC and could significantly reduce the on-target toxicity of Trop2 ADC to normal tissues.

[0559] Table 3 Cell seeding density

[0560] Cell line Number of cells per well (cytotoxicity assay) NCI-H358 2000 NCI-H322 2000 SUM149PT 1000 SCC25 2000 MCF10A 2000

[0561] Example 8: Antitumor efficacy of Trop2 / B7-H3 bispecific ADC molecules in a NCI-H358 mouse xenograft tumor model

[0562] To demonstrate the in vivo efficacy of the Trop2 / B7-H3 bispecific ADC molecule, we used NCI-H358 cells inoculated into CB17-SCID mice to determine the anti-tumor efficacy of the molecule of the present invention. Female mice of SPF grade (14 - 17 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment.

[0563] NCI-H358 cells were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation, and NCI-H358 cells were dispersed with PBS (1X) to prepare a cell suspension with a cell concentration of 2.5x10 7 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB17-SCID mice to establish an NCI-H358 tumor-bearing mouse model. On day 7 after tumor cell inoculation, all mice were randomly divided into 5 groups (5 mice in each group). The dosing dose and method are shown in Table 4. Administration was carried out on day 7 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. As Figure 6A 、 6B shown, the monitoring ended after 50 days.

[0564] On day 50 after inoculation, the relative tumor growth inhibition rate (TGI%) was calculated, and the calculation formula was as follows:

[0565] TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration).

[0566] Tumor volume measurement: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using vernier calipers, and the tumor volume was calculated according to the following formula:

[0567] V = L * W 2 / 2. The body weight was measured using an electronic balance.

[0568] Table 4. Experimental design table

[0569]

[0570] The results of the tumor growth inhibition rate are shown in Table 5 and Figure 6A as follows: On day 50 after inoculation, the tumor growth inhibition rate of a single administration of HZ5C2.9 / hRS7.13-NT3 reached 93%, which was superior to the tumor growth inhibition rates of 29% and 16% of HZ5C2.9-NT3 and hRS7.13-NT3. At the same time, we detected the body weight of the mice, and the results showed that Figure 6B there was no significant difference in the body weight of the mice.

[0571] Table 5. Tumor growth inhibition rate on day 50

[0572]

[0573] Example 9: Antitumor Efficacy of Trop2 / B7-H3 Bispecific ADC Molecule in HCC1806 Mouse Xenograft Tumor Model

[0574] To demonstrate the in vivo efficacy of the Trop2 / B7-H3 bispecific ADC molecule, we used HCC1806 (HCC1806 human breast cancer cells, sourced from ATCC, catalog number CBP60373, Nanjing Kebai Biotechnology Co., Ltd.) cells to inoculate CB17-SCID mice to determine the antitumor efficacy of the molecule of the present invention. Female mice of SPF grade (14 - 17 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment.

[0575] HCC1806 cells were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation, and HCC1806 cells were dispersed with PBS (1X) to prepare a cell suspension with a cell concentration of 2.5x10 7 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB17-SCID mice to establish an HCC1806 tumor-bearing mouse model. On day 7 after tumor cell inoculation, all mice were randomly divided into 5 groups (5 mice per group). The dosing dose and method are shown in Table 6. Administration was carried out on day 7 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. As Figure 7A 、 7B shown, the monitoring ended after 26 days.

[0576] On day 26 after inoculation, the relative tumor growth inhibition rate (TGI%) was calculated, and the calculation formula was as follows:

[0577] TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration).

[0578] Tumor volume measurement: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using vernier calipers, and the tumor volume was calculated according to the following formula:

[0579] V = L * W 2 / 2. Body weight was measured using an electronic balance.

[0580] Table 6. Experimental Design Table

[0581]

[0582]

[0583] The results of the tumor growth inhibition rate are shown in Table 7 and Figure 7AShown as follows: on the 26th day after inoculation, the tumor growth inhibition rate of a single dose of HZ5C2.9 / hRS7.13-NT3 reached 96%, which was superior to the tumor growth inhibition rates of 49% and 46% of HZ5C2.9-NT3 and hRS7.13-NT3, respectively. At the same time, we detected the body weights of the mice, and the results are as Figure 7B shown that there was no significant difference in the body weights of the mice.

[0584] Table 7. Tumor growth inhibition rate on the 26th day

[0585]

[0586] Sequence information:

[0587]

[0588]

[0589]

[0590]

[0591]

Claims

1. An antibody-drug conjugate represented by formula (I): Ab-(L-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, in: Ab is an antibody or a fragment thereof (eg, an antigen-binding fragment) that specifically binds to TROP2 and B7-H3 (eg, human TROP2 and human B7-H3); L is a linker; D is a drug, preferably an anti-tumor compound; and p is an integer selected from 1 to 16, for example 6, 7, 8, 9 or 10, Wherein Ab in formula (I) is a bispecific antibody that specifically binds to B7-H3 and TROP2, which comprises a first antigen binding region that specifically binds to TROP2 and a second antigen binding region that specifically binds to B7-H3, wherein the second antigen binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:2, HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:3, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:7, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:8, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

9.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the second antigen-binding region that specifically binds to B7-H3 comprises VH and VL, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4, and / or the VL comprises or consists of the amino acid sequence of SEQ ID NO:10, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

10.

3. The antibody-drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the second antigen-binding region that specifically binds to B7-H3 comprises VH and VL, wherein VH and VL comprise or consist of the amino acid sequences shown below, respectively: SEQ ID NO: 4 and SEQ ID NO:

10.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the first antigen binding region that specifically binds to TROP2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:19; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:20; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

21.

5. The antibody-drug conjugate of any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, wherein the first antigen-binding region that specifically binds to TROP2 comprises VH and VL, wherein the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the first antigen binding region that specifically binds to TROP2 comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO: 16 and SEQ ID NO:

22.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

8. The antibody-drug conjugate of claim 7 or a pharmaceutically acceptable salt or solvate thereof, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 30 or 31 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98% or 99% or higher identity thereto.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 7 or 8, wherein a mutation that promotes heterodimerization of the first Fc region and the second Fc region of the bispecific antibody is introduced into the first Fc region and the second Fc region.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the mutation in the Fc region is introduced based on the Knob-into-Hole technology, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region, respectively.

11. The antibody-drug conjugate of claim 10, or a pharmaceutically acceptable salt or solvate thereof, wherein one Fc region comprises amino acid substitutions S354C and T366W and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the EU index), e.g., wherein S354C and Y349C form a non-native disulfide bond.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the mutation in the Fc region is introduced based on the Innobody technology.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 12, wherein the Fc region further comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutations.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 7 to 13, wherein one Fc region polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO:27, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO:27, and the other Fc region polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NO:28, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO:

28.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 14, wherein the first antigen-binding region in the bispecific antibody is a Fab fragment, and / or the second antigen-binding region is a Fab.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein the Fab as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein the CH1 comprises (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 32; or (ii) comprises the amino acid sequence of SEQ ID NO: 32 or consists of the amino acid sequence.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 17, wherein the Fab as the first antigen-binding region or the second antigen-binding region comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.

19. The antibody-drug conjugate of claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein the Kappa light chain constant region comprises (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11; or (ii) comprises the amino acid sequence of SEQ ID NO: 11 or consists of the amino acid sequence.

20. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of any one of claims 15 to 19, wherein the first antigen-binding region that specifically binds to TROP2 comprises a first Fab and the second antigen-binding region that specifically binds to B7-H3 comprises a second Fab, wherein the first Fab is connected to the N-terminus of the first Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region), and the second Fab is connected to the N-terminus of the second Fc region at the C-terminus of its CH1 (via or without a linker, such as a hinge region). 21 . The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 20 , wherein the bispecific antibody is an IgG-like antibody having the configuration as shown in FIG. 1 .

22. The antibody-drug conjugate of claim 20 or 21, or a pharmaceutically acceptable salt or solvate thereof, wherein the bispecific antibody comprises a first Fab as a first antigen binding region that specifically binds to TROP2 and a second Fab as a second antigen binding region that specifically binds to B7-H3, wherein the bispecific antibody comprises or consists of: Heavy chain 1: comprising or consisting of the following from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to TROP2 - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to TROP2 - a light chain constant region; Heavy chain 2: comprising or consisting of the following from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to B7-H3 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); Light chain 2: from N-terminus to C-terminus, it comprises the following: a light chain variable region of a Fab that specifically binds to B7-H3 - a light chain constant region, Preferably, each domain is directly linked; Optionally, the first Fc region comprises mutations Y349C, T366S, L368A and Y407V, and the second Fc region comprises mutations S354C and T366W, or vice versa; optionally, the first Fc region and the second Fc region further comprise L234A / L235A mutations, respectively.

23. The antibody-drug conjugate of claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein heavy chain 1 comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; light chain 1 comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; heavy chain 2 comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consists of said amino acid sequence; and light chain 2 comprises the amino acid sequence of SEQ ID NO:

7. The amino acid sequence shown in NO:12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, or consisting of the amino acid sequence.

24. The antibody-drug conjugate according to any one of claims 1 to 23, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is a cytotoxic agent, such as a camptothecin, an auristatin, a maytansine, a taxane, an anthracycline, a vinca alkaloid, a MEK inhibitor or a KSP inhibitor.

25. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 24, wherein D is represented by formula (D-1): Where R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2; R 4 Selected from H or C1-C4 alkyl.

26. The antibody-drug conjugate according to claim 25, or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 For H, R 2 is a C1-C6 alkyl group, and R 3 is -F.

27. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein D is represented by formula (D-2): Where R 1 , R 2 and R 3 As defined in claim 25 or 26.

28. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein D is represented by formula (D-3) or formula (D-4):

29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 28, wherein -L- is the following structure: -Z-E-NH-CH2-Q-L 2 -L 1 - Where Z is connected to Ab, L 1 Connect with D; Z is selected from wherein m is an integer of 1 to 10; the carbonyl group at the right end of Z is covalently linked to E; E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z; Q is -O- or -S-; L 1 is not present or -(C1-C 10 Alkylene)-; L 2 Does not exist, -N(R 5 )C(O)-(C1-C 10 Alkylene)-* or -C(O)N(R 5 )-(C1-C 10 Alkylene)-*; wherein * indicates that the terminus is covalently linked to Q; and R 5 It is H or C1-C6 alkyl.

30. The antibody-drug conjugate according to claim 29 or a pharmaceutically acceptable salt or solvate thereof, wherein E is a peptide residue consisting of 2, 3 or 4 amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group; and -L 2 -L 1 - is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C 10 Alkylene)-*, wherein * indicates that the terminus is covalently linked to Q; R 5 It is H or C1-C6 alkyl.

31. The antibody-drug conjugate according to claim 30 or a pharmaceutically acceptable salt or solvate thereof, in, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala- or Where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N-terminus; and -L 2 -L 1 - is -(C1-C6 alkylene)-.

32. The antibody-drug conjugate according to claim 31 or a pharmaceutically acceptable salt or solvate thereof, Among them -ZE-NH-CH2-QL 2 -L 1 - is the following structure Its right end is connected to D.

33. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 32, wherein the antibody-drug conjugate of formula (I) has the following structure: Ab’-(S-L-D) p (I’) wherein Ab' is as defined in any one of claims 1-23 for Ab, and L, D and p are as defined in any one of claims 1-32.

34. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 33, having an average DAR value of 5-11, such as 7.5-8.

5.

35. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is as shown in the following structure: Wherein Ab is a bispecific antibody that specifically binds to TROP2 and B7-H3, and the bispecific antibody comprises heavy chain 1, light chain 1, heavy chain 2 and light chain 2, or is composed of the four chains, wherein Heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23; heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6; and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; q is as defined in claim 1 for p, Preferably, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR of 5-11 or 7.5-8.

5.

36. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 35 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

37. A pharmaceutical combination comprising the antibody-drug conjugate of any one of claims 1 to 35 or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

38. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 35, or a pharmaceutical composition according to claim 36, or a pharmaceutical combination according to claim 37.

39. The method of claim 38, wherein the tumor is a cancer, preferably the cancer has elevated levels (e.g., nucleic acid or protein levels) of B7-H3 and / or TROP2, e.g., compared to corresponding tissues of healthy individuals or healthy tissues adjacent to the patient's cancer tissue.

40. The method of claim 39, wherein the cancer is selected from lung cancer, such as non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, kidney cancer, bladder cancer, ovarian cancer, or pancreatic cancer.

41. The method of any one of claims 38-40, wherein the method further comprises administering to the subject one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modalities comprise radiation therapy or surgery, or the therapeutic agents comprise chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

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