Multi-specific antibody targeting her3 and trop2 and conjugate thereof

Abstract

Provided in the present application are an HER3 single-domain antibody, a multi-specific antigen-binding protein and protein-drug conjugate comprising the single-domain antibody, and the use thereof in the preparation of a drug for treating / preventing tumors.

Description

Multispecific antibodies and conjugates targeting HER3 and TROP2 Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to multispecific antibodies and conjugates thereof targeting HER3 and TROP2. Background Art

[0002] Cancer has become the second largest health threat in the world, and chemotherapy based on cytotoxic drugs has always been the main means of cancer treatment. These cytotoxic drugs include DNA base analogs (such as 5-fluorouracil and 8-azaguanine), DNA interaction agents (such as cisplatin and actinomycin D), antimetabolites (such as aminopterin and methotrexate) and microtubule inhibitors (such as paclitaxel and vincristine derivatives), etc. Most chemotherapy drugs have a relatively low therapeutic index, and the serious side effects are mainly caused by off-target effects. To solve this problem, scientists have been exploring cancer treatments with a higher therapeutic window.

[0003] As early as the early 20th century, Paul Ehrlich first proposed the concept of a "magic bullet," hypothesizing that certain compounds could directly enter target tumor cells. Theoretically, these compounds could effectively kill cancer cells while being harmless to normal cells. One possible approach is to identify markers that can distinguish tumor cells from normal cells, such as the HER2 molecule on the surface of breast cancer cells and the CD20 molecule on the surface of B-cell lymphomas. Since the advent of hybridoma technology in 1975, an increasing number of monoclonal antibodies targeting these tumor antigens have been developed. Although monoclonal antibody drugs have relatively good targeting, their effectiveness in killing tumor cells is far inferior to that of chemotherapy. Therefore, a new concept, antibody-drug conjugates (ADCs), has emerged, aiming to bridge antibodies and cytotoxic substances through a linker to increase the tumor therapeutic window.

[0004] Human trophoblast cell surface antigen 2 (TROP2) is a single-pass transmembrane glycoprotein belonging to the GA733 gene family. It is primarily expressed in epithelial cells and regulates intracellular calcium levels. Evidence suggests that TROP2 influences intracellular signaling pathways responsible for cell growth, proliferation, and transformation. During embryonic development, TROP2 protein plays a crucial role in placental formation, embryo implantation, stem cell proliferation, and organ development. Evidence suggests that TROP2 expression is upregulated in many tumor cells, promoting tumor growth, proliferation, and invasion. TROP2 is widely expressed in solid tumors. One study demonstrated that 100% of squamous cell carcinomas (n=72) and 93.9% of adenocarcinomas (n=181) overexpressed TROP2. Its expression is implicated in signaling pathways involved in tumor proliferation, migration, invasion, and metastasis. Therefore, high expression of Trop-2 is an adverse prognostic factor affecting the overall survival of various solid tumors, including breast cancer. At the same time, based on the fact that the Trop-2 expression rate in breast cancer can reach 78%, while in HR+ / HER2- breast cancer it is as high as 78.5%, and in TNBC it can even be as high as 95%, TROP2 has naturally become the "hottest target" for breast cancer after HER2.

[0005] Human epidermal growth factor receptor 3 (HER3), also known as ErbB3, is a cell-surface receptor tyrosine kinase. It is a member of the ErbB family, which includes EGFR (ErbB1), HER2 / neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Discovered in 1989, this member of the ERBB family remains unapproved for over three decades. HER3 overexpression is common in various cancers. Unlike other HER family members, HER3 lacks intracellular kinase activity but can phosphorylate tyrosine kinases by forming heterodimers with other receptors, such as HER2. Ligand-induced receptor dimerization enables intracellular signaling through PI3K / AKT and MAPK / ERK signaling, playing a critical role in cell survival and proliferation, ultimately leading to tumor progression. Recent studies have shown that HER3 overexpression may be associated with a poor prognosis in many solid tumors, including breast, gastric, ovarian, and melanoma.

[0006] Currently, there are no multispecific antibodies or antigen-binding fragments that simultaneously target HER3 and TROP2, nor are there any ADC drugs that simultaneously target these two targets. Summary of the Invention

[0007] In a first aspect, the present invention relates to a human epidermal growth factor receptor 3 (HER3) binding protein comprising at least one immunoglobulin single variable domain, such as a VHH derived from a camelid.

[0008] In a second aspect, the present invention relates to a multispecific antigen-binding protein comprising an antibody or antigen-binding fragment that binds to trophoblast cell surface antigen 2 (TROP2) and at least one immunoglobulin single variable domain that binds to human epidermal growth factor receptor 3 (HER3).

[0009] In a third aspect, the present invention also relates to an antibody-drug conjugate composed of a multispecific antigen-binding protein that binds to HER3 and TROP2 and a cytotoxin, and its anti-tumor effect.

[0010] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings are briefly described as follows:

[0012] FIG1 shows the affinity results of iBT11 and HER3 (ELISA method); □ represents iBT11-Chis, and ○ represents iBT1-Chis.

[0013] Figure 2 shows the MS comparison results of the conjugated product Antibody A-ADC and Antibody A naked antibody.

[0014] FIG3 shows the overall reaction formula for preparing Antibody A-ADC from Antibody A.

[0015] FIG4 shows the killing effect of antibody A-ADC on BxPC-3 cells.

[0016] FIG5 shows the killing effect of antibody A-ADC on A431 cells.

[0017] FIG6 shows the killing effect of antibody A-ADC on CAPAN-2 cells.

[0018] FIG7 shows the killing effect of antibody A-ADC on HCC827 cells.

[0019] FIG8 shows the inhibition rate of antibody A-ADC on the proliferation of BxPC-3 and 293T-GFP cells.

[0020] FIG9 shows the tumor volume change curve in the HCC827 CDX model under the action of different doses of antibody A-ADC.

[0021] Figure 10 shows the tumor volume change curve in the NCI-N87 CDX model under the action of different doses of antibody A-ADC.

[0022] FIG11 shows the tumor volume change curve in the osimertinib-resistant human lung cancer PDX model under the action of different doses of antibody A-ADC.

[0023] Figure 12 shows the tumor volume change curves in the HCC827 CDX model under the action of antibody A-ADC and different doses of positive control drugs. The arrows marked on the horizontal axis (time axis) indicate the specific time points of the four drug administrations. DETAILED DESCRIPTION

[0024] Definition of terms

[0025] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0026] The terms "complete antibody," "full-length antibody," or "whole antibody" are used interchangeably herein and generally refer to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions can be connected by a "J" region of about 12 or more amino acids, and the heavy chain can also include a "D" region of about 3 or more amino acids. The heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can be composed of three domains (CH1, CH2, and CH3). The light chain can be composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region can be composed of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For example, VH and VL can each comprise or consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site.

[0027] The term "antibody fragment" or "antigen-binding fragment" refers to a molecule that is different from a full-length antibody, which comprises a portion of a full-length antibody and retains the ability to specifically bind to an antigen (i.e., binds to the same antigen as the full-length antibody from which the portion or fragment is derived). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent antibodies or fragments thereof; camelid antibodies (heavy chain antibodies). The term also encompasses fragments of bispecific or multispecific antibodies, and / or bispecific or multispecific antibodies formed from antibody fragments.

[0028] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies 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, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0029] In the present invention, the regions of CDR defined by Kabat, AbM or IMGT schemes are as follows:

[0030] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0031] Herein, when referring to "Kabat CDR", it refers to the CDR determined based on the Kabat scheme. Similarly, "Chothia CDR" refers to the CDR determined based on the Chothia scheme, "Abm CDR" refers to the CDR determined based on the Abm scheme, and "IMGT CDR" refers to the CDR determined based on the IMGT scheme.

[0032] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0033] As used herein, the terms "antigen binding protein", "binding protein" or "binding molecule" include molecules containing at least one antigen binding site, wherein the site specifically binds to a target molecule. Antigen binding proteins can be antibodies, such as full-length antibodies, or antigen-binding fragments of antibodies, or chimeric antigen receptors (CARs), or any other polypeptides with higher than usual affinity, such as scaffold proteins, cyclic peptides, soluble receptors, receptor-antibody (Rab) proteins or fragments of these polypeptides. The target molecule can be any molecule, and in particular herein can be a conformational epitope of HER3, TROP2, especially HER3, TROP2.

[0034] As used herein, the term "multispecific antigen-binding protein" is used in the broadest sense to refer to a binding protein that can bind (preferably specifically bind) two or more antigens. Without being bound by theory, in general, a multispecific antigen-binding protein comprises a plurality of relatively independent structures that each provide binding specificity for the plurality of antigens, for example, the plurality of structures each independently comprise one or more binding sites for each target antigen, for example, at least one of the plurality of structures is an antibody or antibody fragment. In some aspects, at least one binding specificity is provided by an antibody. In some aspects, at least one binding specificity is provided by a full-length antibody. In some aspects, at least one binding specificity is provided by a VHH domain. In some aspects, the multispecific binding protein is a bispecific antibody.

[0035] In this application, the term "bispecific antibody" generally refers to an antibody that can bind to two antigens or antigenic epitopes respectively. In some embodiments of the present invention, the bispecific antibody may include a light chain and a heavy chain of an antibody that can specifically bind to a first antigen or antigenic epitope, and a light chain and a heavy chain of an antibody that can specifically bind to a second antigen or antigenic epitope. In some embodiments of the present invention, the bispecific antibody may include a VHH domain that can specifically bind to a first antigen or antigenic epitope, and a light chain and a heavy chain of an antibody that can specifically bind to a second antigen or antigenic epitope.

[0036] The term "epitope" or "antigenic epitope" generally refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across separate protein amino acid residues.

[0037] The term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein can bind to. Specificity can be determined based on the affinity and / or avidity of an antigen binding protein. Avidity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and a related antigen. Avidity is related to both the affinity between the antigen binding site on its antigen binding protein and the number of related binding sites present on the antigen binding protein.

[0038] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without losing the rest of the protein and / or the function of the domain.

[0039] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide that essentially consists of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0040] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, and three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively, that separate the framework regions. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site. The variable domains of the heavy and light chains of a natural antibody typically have similar structures.

[0041] The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that can specifically bind to an antigen epitope without being paired with other immunoglobulin variable domains. A single heavy chain variable (VH) or light chain variable (VL) domain can be sufficient to provide antigen binding specificity. An example of an immunoglobulin single variable domain in the meaning of the present application is a "domain antibody", such as an immunoglobulin single variable domain VH or VL (VH domain or VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae as defined below, sometimes also referred to as a "single domain antibody", or a "nanobody".

[0042] "VHH domain", also known as single-domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish such variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to as "VH domains" in this application). The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional four-chain antibodies, in which the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain. VHH is, for example, from a camelid, such as an alpaca, or a humanized form or sequence-optimized form thereof (e.g., an affinity-matured form to increase binding affinity). In some embodiments, VHH is a monovalent, monospecific polypeptide molecule consisting of or essentially consisting of a single heavy chain variable region (e.g., a heavy chain variable region of a heavy chain antibody). Preferably, in one embodiment, the present invention provides an immunoglobulin single variable domain comprising a humanized VHH. Preferably, in one embodiment, the present invention provides an antibody comprising a humanized VHH. Preferably, in one embodiment, the present invention provides a multispecific antigen-binding protein (e.g., a multispecific antibody) comprising a humanized VHH.

[0043] In the context of this application, the terms "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0044] For example, as shown in Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999) (see Figure 2 thereof), the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0045] Alternative methods for numbering the amino acid residues of VH domains are known in the art and can be similarly applied to VHH domains. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures.

[0046] Single domain antibodies or VHHs can also be contained in larger polypeptides / proteins. Examples of polypeptides / proteins containing the VHHs of the present invention include, but are not limited to, heavy chain antibodies (HcAbs).

[0047] The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, it may comprise VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from the N segment to the C segment, or may comprise VH-CH1-CH2-CH3. The heavy chain antibody of the present invention may also encompass homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may comprise VH from a standard antibody or VH from a single domain antibody. For example, the VH in the heavy chain antibody may be VHH. For example, the heavy chain antibody may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), a humanized form thereof or a sequence-optimized form thereof (affinity matured form), or a fragment thereof (e.g., a fragment comprising at least a portion of the constant region). The heavy chain antibody may also encompass an antibody formed by fusing a heavy chain variable region or VHH with an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region).

[0048] The term "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. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In some embodiments, the Fc region of the present invention is from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 2 or 3, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0049] The term "protein-drug conjugate" generally refers to a binding protein (e.g., an antibody or its antigen-binding fragment) connected to one or more chemical drugs, such as antibody-drug conjugates (ADC). The chemical drug can be any therapeutic agent and / or cytotoxic agent. The antibody-drug conjugate can have any number of drugs coupled to the antibody from 1 to 16, for example, 2, 4, 6 or 8 drug-loaded species can be included. In the present invention, the drug can include mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic protective agents, hormones, anti-hormonal agents, corticosteroids, photosensitizers, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and / or radiosensitizers, etc.

[0050] In this application, the term "drug / antibody ratio" or "DAR" generally refers to the number of drugs attached to the antibody (or protein) of an ADC. The DAR of an ADC can range from 1-8, or even higher (e.g., 10), and the range of DAR can depend on the number of attachment sites on the antibody. In this application, the DAR can be the number of drugs loaded onto a single antibody. The DAR can also be the average or mean DAR of a group of ADCs.

[0051] The term "linker" refers to a structural fragment that connects a drug (e.g., a small molecule drug) to an antibody portion. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or antigen-binding fragment thereof before being attached to the antibody or antigen-binding fragment thereof.

[0052] In this application, the term "HER3" generally refers to human epidermal growth factor receptor 3 (SwissProt P21860). Human HER3 is also known as ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, and is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4. HER3 is a transmembrane receptor similar to EGFR, consisting of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a C-terminal phosphorylation domain. HER3 is able to bind to its ligand, and although it cannot directly transmit signals into cells via protein phosphorylation, it can indirectly activate signal transduction pathways by forming heterodimers with other HER family members that have kinase activity. Dimer formation between HER family members expands the signal transduction potential of HER3 and is not only a means of signal diversification but also a means of signal amplification. The HER3 may include any variants, truncations, fragments, isoforms, and species homologs of HER3 that are naturally expressed by cells, including tumor cells, or expressed by cells transfected with a HER3 gene or cDNA.

[0053] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.

[0054] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0055] The term "cycloalkylene" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon radical derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane.

[0056] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon.

[0057] The term "heterocycloalkylene" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m A residue derived from an alkane radical comprising a ring containing at least one heteroatom (wherein m is an integer from 0 to 2) but not including the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms being carbon, and being derived from a residue derived from an alkane radical by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of the parent alkane radical.

[0058] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.

[0059] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring. The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0060] The term "arylene" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring having a conjugated π electron system, and the residue is derived by removing two hydrogen atoms from two different carbon atoms of a parent aromatic ring.

[0061] The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0062] The term "heteroarylene" refers to a heteroaromatic polycyclic ring containing 1 to 4 heteroatoms, 5 to 14 ring atoms, and derived from a parent aromatic ring by removing two hydrogen atoms from two different carbon atoms.

[0063] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the 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 unsubstituted.

[0064] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effects and properties of the conjugates of the present invention and are not biologically or otherwise undesirable. The conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, pharmaceutically acceptable, non-toxic acid addition salts refer to salts formed between the conjugates of the present invention and organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, and malic acid. Pharmaceutically acceptable, non-toxic base addition salts refer to salts formed between the conjugates of the present invention and organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium, or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing an N group.

[0065] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0066] As used herein, the term "and / or" refers to any one of the alternatives or two or more of the alternatives.

[0067] As used herein, the term "comprises" or "comprising" means including the elements, integers or steps mentioned, but not excluding any other elements, integers or steps. In this article, when the term "comprises" or "comprising" is used, unless otherwise indicated, combinations of the elements, integers or steps mentioned are also covered.

[0068] The term "administering" generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0069] In this application, the term "about" generally refers to variation within a range of 0.5%-10% above or below a specified value, for example, variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Unless otherwise specified, the numerical values ​​mentioned in this application are considered to be modified by "about". If in doubt, or if the error range for a particular value or parameter is not commonly understood in the art, "about" means ±5% of that value or parameter.

[0070] The term "effective amount" refers to an amount or dosage of an antibody, fragment, composition, or combination of the present invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both a "therapeutically effective amount" and a "prophylactically effective amount" may be included.

[0071] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome, at the dosage and for the period of time required. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" is an amount effective to achieve the desired preventive outcome, at the dosage and for the period of time required. Typically, because a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0072] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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.

[0073] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages or be metastatic.

[0074] 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.

[0075] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0076] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0077] As used herein, "treat," ...

[0078] Detailed Description of the Invention

[0079] Human epidermal growth factor receptor 3 (HER3) binding protein

[0080] In one aspect, the present application provides a single-domain antibody against human epidermal growth factor receptor 3 (HER3), comprising CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1.

[0081] In some embodiments, the CDR1, CDR2, and CDR3 are defined according to the following schemes: Kabat, AbM, Chothia, or IMGT.

[0082] In some embodiments, the single domain antibody is camelid, humanized, or chimeric.

[0083] In some embodiments, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 8-10 (Kabat scheme), SEQ ID NO: 11-13 (AbM scheme), SEQ ID NO: 14-16 (Chothia scheme) and SEQ ID NO: 17-19 (IMGT scheme).

[0084] In some embodiments, the single-domain antibody comprises (1) SEQ ID NO: 1; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, compared to SEQ ID NO: 1, or consists of an amino acid sequence.

[0085] In some embodiments, the single domain antibody is humanized.

[0086] In some embodiments, the single-domain antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-7.

[0087] In some embodiments, the single-domain antibody consists of the amino acid sequence shown in any one of SEQ ID NOs: 2-7.

[0088] On the other hand, the present application provides a human epidermal growth factor receptor 3 (HER3) binding protein, which comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1.

[0089] The "at least one immunoglobulin single variable domain" may be one or more. Unless otherwise specified, the amino acid sequences of the multiple immunoglobulin single variable domains may be the same or different. In some embodiments, "multiple" may be 2, 3, 4, 5, or more.

[0090] In some embodiments, the CDR1, CDR2, and CDR3 are defined according to the following schemes: Kabat, AbM, Chothia, or IMGT.

[0091] In some embodiments, the immunoglobulin single variable domain is camelid, humanized, or chimeric.

[0092] In some embodiments, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 8-10 (Kabat scheme), SEQ ID NO: 11-13 (AbM scheme), SEQ ID NO: 14-16 (Chothia scheme) and SEQ ID NO: 17-19 (IMGT scheme).

[0093] In some embodiments, the at least one immunoglobulin single variable domain comprises, or consists of, one or more VHHs, wherein at least one VHH is as shown in SEQ ID NO: 1.

[0094] In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of (1) SEQ ID NO: 1; (2) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1; or (3) a VHH represented by an amino acid sequence comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO: 1, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0095] In some embodiments, the at least one immunoglobulin single variable domain consists of one or more VHHs set forth in an amino acid sequence that (1) is SEQ ID NO: 1; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, compared to SEQ ID NO: 1.

[0096] In some embodiments, the immunoglobulin single variable domain is a humanized VHH, comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the humanized VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO: 1. For example, the amino acid sequence of the humanized immunoglobulin single variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 1.

[0097] In some embodiments, the at least one immunoglobulin single variable domain may comprise one or more humanized VHHs as described above.

[0098] In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences shown in SEQ ID NOs: 2-7.

[0099] In some embodiments, the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences shown in SEQ ID NOs: 1-7.

[0100] In some embodiments, the at least one immunoglobulin single variable domain consists of one or more amino acid sequences shown in SEQ ID NOs: 1-7.

[0101] In other embodiments, the at least one immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. For example, the at least one immunoglobulin single variable domain comprises one or more (1) SEQ ID NO: 1; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO: 1. The at least one immunoglobulin single variable domain comprises one or more (1) SEQ ID NO: 2; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2; or (3) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2. NO:2 comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, a VHH set forth in an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions; said at least one immunoglobulin single variable domain comprises one or more of (1) SEQ ID NO:3; (2) having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:3; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, a VHH set forth in an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO:3; said at least one immunoglobulin single variable domain comprises one or more of (1) SEQ ID NO:4; (2) having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:3; NO:4 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO:4; the at least one immunoglobulin single variable domain comprises one or more (1) SEQ ID NO:5; (2) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:5;or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, a VHH represented by an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO: 5; said at least one immunoglobulin single variable domain comprises one or more (1) SEQ ID NO: 6; (2) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 6; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, a VHH represented by an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO: 6; or, said at least one immunoglobulin single variable domain comprises one or more (1) SEQ ID NO: 7; (2) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 6. NO:7 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity; or (3) an amino acid sequence comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO:7.

[0102] For example, in some embodiments, the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 1; the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 2; the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 3; the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 4; the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 5; the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 6; or, the at least one immunoglobulin single variable domain consists of one or more VHHs shown in SEQ ID NO: 7.

[0103] In some embodiments, the HER3 binding protein comprises one of the aforementioned immunoglobulin single variable domains.

[0104] In some embodiments, the HER3 binding protein comprises 2, 3, 4, or 5 or more of the aforementioned immunoglobulin single variable domains.

[0105] In some embodiments, the HER3-binding proteins of the present application, in addition to at least one immunoglobulin single variable domain, further comprise an immunoglobulin Fc region. Inclusion of an immunoglobulin Fc region in the HER3-binding proteins of the present disclosure can allow the binding molecules to form dimers. Fc regions useful in the present disclosure can be derived from immunoglobulins of different subtypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

[0106] In some embodiments, mutations can be introduced into the wild-type Fc sequence to alter Fc-mediated related activities. Such mutations include, but are not limited to: a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter FcRn-mediated in vivo half-life. Such mutations are described in the following literature: Leonard G Presta, Current Opinion in Immunology 2008, 20:460–470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al., J Immunol, 2006, 176:346-356. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the CH2 region can be mutated to increase or remove Fc-mediated ADCC or CDC activity or to enhance or reduce FcRn affinity. In addition, the stability of the protein can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the hinge region.

[0107] The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 20.

[0108] In some specific embodiments, in the HER3-binding protein of the present application, the immunoglobulin Fc region (eg, the Fc region of human IgG1) is directly or indirectly linked via a linker to the C-terminus of the immunoglobulin single variable domain (eg, VHH).

[0109] In some embodiments, the HER3 binding protein of the present application has at least one of the following characteristics:

[0110] (a) specifically binds to human HER3;

[0111] (b) K binding to human HER3 D Values ​​less than 1x10 -7 M, preferably less than 1x10 -8 M;

[0112] (c) ECs that specifically bind to human HER3 50 The value is less than 200 ng / mL, preferably less than 100 ng / mL, more preferably less than 50 ng / mL;

[0113] (d) can block the formation of heterodimers between HER3 and HER2;

[0114] (e) capable of blocking the binding of HER3 to its ligand (e.g., NRG1 or NRG1b1), for example, with an IC of no more than 200 ng / mL, preferably no more than 150 ng / mL, more preferably no more than 120 ng / mL 50 The value blocks the binding of HER3 to its ligand;

[0115] (f) Can inhibit the proliferation of tumor cells.

[0116] Fusion protein

[0117] On the other hand, the present application also relates to a fusion protein comprising the HER3-binding protein or the single-domain antibody against HER3 described in the present disclosure.

[0118] In some specific embodiments, the fusion protein is a multispecific antigen-binding protein, e.g., a bispecific antigen-binding protein, e.g., a bispecific antibody. In some more specific embodiments, the multispecific antigen-binding protein further comprises a second binding specificity and optionally further binding specificities.

[0119] In some embodiments, in addition to the HER3-binding protein or single-domain antibody directed against HER3, the fusion protein further comprises one or more other biologically active proteins. The biologically active proteins can be any proteins with biological, therapeutic, preventive, or diagnostic significance or function, which, when administered to a subject, mediate biological activity that can prevent or alleviate a disease, disorder, or condition. Specifically, the biologically active proteins can be agonists, antagonists, modulators, ligands, cytokines, enzymes, or hormones, and are particularly useful for preventing and / or treating tumors, particularly solid tumors and / or non-solid tumors.

[0120] Multispecific antigen-binding proteins

[0121] On the other hand, the present application also relates to a multispecific antigen-binding protein, which comprises a structural portion that specifically binds to HER3 (HER3 binding region), for example, a HER3 binding protein, preferably, the HER3 binding protein described in the present application.

[0122] In some embodiments, the multispecific antigen-binding protein comprises, in addition to the HER3-binding protein, one or more additional antigen-binding regions, which provide different antigen-binding specificities, i.e., bind to a different antigen or a different epitope of the same antigen as the HER3-binding protein.

[0123] In some embodiments, the multispecific antigen-binding protein comprises a trophoblast cell surface antigen 2 (TROP2) binding region and a human epidermal growth factor receptor 3 (HER3) binding region. In some more specific embodiments, the trophoblast cell surface antigen 2 (TROP2) binding region and the human epidermal growth factor receptor 3 (HER3) binding region are both antibodies or antibody fragments (e.g., immunoglobulin single variable domains), i.e., the binding specificity for TROP2 and HER3 are both provided by antibodies or antibody fragments, i.e., the multispecific antigen-binding protein is an anti-TROP2×HER3 bispecific antibody.

[0124] In some embodiments, the multispecific antigen-binding protein comprises a trophoblast cell surface antigen 2 (TROP2) binding protein, e.g., an antibody or antigen-binding fragment thereof that binds TROP2, and at least one immunoglobulin single variable domain that binds human epidermal growth factor receptor 3 (HER3) as described above;

[0125] Specifically, the antibody or antigen-binding fragment thereof that binds to TROP2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1, a VH CDR2, and a VH CDR3 selected from any one group of SEQ ID NOs: 22-24 (Kabat scheme), SEQ ID NOs: 25-27 (AbM scheme), SEQ ID NOs: 28-30 (Chothia scheme), and SEQ ID NOs: 31-33 (IMGT scheme),

[0126] The light chain variable region comprises a VL CDR1, a VL CDR2, and a VL CDR3 selected from any one group consisting of SEQ ID NOs: 35-37 (Kabat scheme), SEQ ID NOs: 38-40 (AbM scheme), SEQ ID NOs: 41-43 (Chothia scheme), and SEQ ID NOs: 44-46 (IMGT scheme).

[0127] In some embodiments, in the multispecific antigen-binding protein, the HER3-binding immunoglobulin single variable domain comprises a VHH CDR1, a VHH CDR2, and a VHH CDR3 selected from any one group consisting of SEQ ID NOs: 8-10 (Kabat scheme), SEQ ID NOs: 11-13 (AbM scheme), SEQ ID NOs: 14-16 (Chothia scheme), and SEQ ID NOs: 17-19 (IMGT scheme).

[0128] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises (1) SEQ ID NO: 21; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 21; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO: 21, or consists of the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

[0129] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises (1) SEQ ID NO: 34; (2) is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 34; or (3) comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to SEQ ID NO: 34, or consists of the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34.

[0130] In some embodiments, the heavy chain of the antibody or antigen-binding fragment that binds to TROP2 further comprises the constant region of human IgG1 or a variant thereof, for example, comprising (1) SEQ ID NO: 47; (2) having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 47; or (3) comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO: 47, for example, an amino acid sequence shown in an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0131] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof that binds to TROP2 further comprises the constant region of human Igκ or a variant thereof, for example, comprising (1) SEQ ID NO: 48; (2) having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 48; or (3) comprising one or more amino acid substitutions compared to SEQ ID NO: 48, preferably conservative amino acid substitutions, for example, an amino acid sequence shown in an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0132] In some specific embodiments, the heavy chain of the antibody that binds to TROP2 comprises (1) SEQ ID NO: 49; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, compared to SEQ ID NO: 49. In some specific embodiments, the heavy chain of the antibody that binds to TROP2 comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0133] In some specific embodiments, the light chain of the antibody that binds to TROP2 comprises (1) SEQ ID NO: 50; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, compared to SEQ ID NO: 50. In some specific embodiments, the light chain of the antibody that binds to TROP2 comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0134] As an example, the antibody that binds to TROP2 may have the same structure as Sacituzumab (hRS7), which is published in WHO Drug Information Proposed INN List 115 (2016).

[0135] In some embodiments, in the multispecific antigen-binding protein, the at least one immunoglobulin single variable domain that binds HER3 comprises (1) one of SEQ ID NOs: 1-7; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 1-7; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to one of SEQ ID NOs: 1-7, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.

[0136] In some embodiments, the at least one immunoglobulin single variable domain that binds HER3 comprises one or more of (1) SEQ ID NO: 1; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO: 1, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the at least one immunoglobulin single variable domain that binds HER3 comprises one or more of (1) SEQ ID NO:7; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to SEQ ID NO:7, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.

[0137] In some embodiments, the multispecific antigen-binding protein comprises one of the aforementioned immunoglobulin single variable domains; in other embodiments, the multispecific antigen-binding protein comprises two of the aforementioned immunoglobulin single variable domains.

[0138] In some embodiments, in the multispecific antigen-binding protein, the at least one immunoglobulin single variable domain that binds HER3 is directly or indirectly linked via a linker to an antibody or antigen-binding fragment that binds TROP2.

[0139] In some embodiments, the linker is a polypeptide, for example, a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. For example, the linker is a flexible linker, such as one or more of (GGG)n, (GGGS)n, (GGGA)n, (GGGAA)n, (GGGGS)n, and (GGGGGS)n, wherein n is an integer selected from 1 to 30, an integer selected from 1 to 20, an integer selected from 1 to 10, an integer selected from 1 to 9, an integer selected from 1 to 8, an integer selected from 1 to 7, an integer selected from 1 to 6, an integer selected from 1 to 5, an integer selected from 1 to 4, or an integer selected from 1 to 3. In some embodiments, the linker is GGGGGS, GGGGS, GS, GAP, (GGGGS)×3, GAPGGGGGS, etc.

[0140] In some embodiments, the C-terminus of the at least one immunoglobulin single variable domain that binds HER3 is linked to the N-terminus of the heavy chain of the antibody or antigen-binding fragment that binds TROP2.

[0141] In some embodiments, the N-terminus of the at least one immunoglobulin single variable domain that binds HER3 is linked to the C-terminus of the heavy chain of the antibody or antigen-binding fragment that binds TROP2.

[0142] In some embodiments, the C-terminus of the at least one immunoglobulin single variable domain that binds HER3 is linked to the N-terminus of the light chain of the antibody or antigen-binding fragment that binds TROP2.

[0143] In some embodiments, the N-terminus of the at least one immunoglobulin single variable domain that binds HER3 is linked to the C-terminus of the light chain of the antibody or antigen-binding fragment that binds TROP2.

[0144] In some specific embodiments, the multispecific antigen-binding protein comprises a first polypeptide represented by SEQ ID NO:51 and a second polypeptide represented by SEQ ID NO:50.

[0145] In some specific embodiments, the multispecific antigen-binding protein comprises more than one first polypeptide of SEQ ID NO: 51 and more than one second polypeptide of SEQ ID NO: 50. In some embodiments, the multispecific antigen-binding protein forms homodimers through disulfide bonds in the hinge region of the first polypeptides.

[0146] In some embodiments, the multispecific antigen binding protein described herein can (a) simultaneously bind to HER3 and TROP2 (ELISA method), for example, its EC 50 A value of <100 nM, preferably <50 nM; (b) binding to cells expressing HER3 or TROP2; and / or (c) bridging cells expressing TROP2 and HER3.

[0147] Protein-drug conjugates

[0148] On the other hand, the present application also relates to a protein-drug conjugate having a structure of Formula I:

[0149] P-(L1-sp1-L2-sp2-D)n(I),

[0150] Wherein, protein P comprises the multispecific antigen-binding protein described in the present application, D is a biologically active substance, L1 is a linker for connecting to P, sp1 is a first spacer unit, L2 is a cleavable linker, sp2 is a second spacer unit and is connected to D, and n=1 to 20.

[0151] In some embodiments, L1 is selected from:

[0152] (the side connected to the protein is marked as P, and the side connected to the first spacer unit is marked as sp1), wherein Ar represents C 6-10 arylene, which is optionally substituted by halogen, C 1-6 Alkyl substituted; R1 is selected from hydrogen, halogen and C 1-6 Alkyl; Z is selected from a straight bond, C 2-6 Alkynylidene, C 2-6 Alkenylene, C 6-10 Arylene, 5-10 membered heteroarylene, amide, sulfonamide, imine and CF2.

[0153] In some embodiments, L1 is selected from

[0154] In some embodiments, the structure of the first spacer unit sp1 is as shown in Formula II:

[0155] (The side connected to L1 is labeled L1, and the side connected to L2 is labeled L2), wherein a1=0 or 1, a2=an integer from 0 to 6, b1=0 or 1, b2=an integer from 0 to 16, b3=an integer from 0 to 16, c=an integer from 0 to 6, and at least one of b2 and b3 is 0.

[0156] In some embodiments, in the structure of sp1, a1=1; in other embodiments, a1=0.

[0157] In some embodiments, a2=0, 1, 2, 3, 4, 5, or 6.

[0158] In some embodiments, b1=0 or 1.

[0159] In some embodiments, b2=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0160] In some embodiments, b3=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0161] In some embodiments, c=0, 1, 2, 3, 4, 5, or 6.

[0162] The above options a1, a2, b1, b2, b3 and c can be combined arbitrarily, provided that at least one of b2 and b3 is 0.

[0163] In some specific embodiments, the structure of sp1 is selected from the following group:

[0164] (1) a1=0, a2=2, 3, 4, 5 or 6, b1=0, b2=0, b3=0, c=0;

[0165] (2) a1=0, a2=0, b1=0, b2=0, b3=0, c=2, 3, 4, 5, or 6;

[0166] (3) a1=1, a2=2, 3, 4, 5, or 6, b1=1, b2=2, 3, 4, 5, 6, 7, or 8, b3=0, c=0;

[0167] (4) a1=0, a2=2, 3, 4, 5, or 6, b1=1, b2=2, 3, 4, 5, 6, 7, or 8, b3=0, c=0; and

[0168] (5) a1=1, a2=0, b1=0, b2=0, b3=2, 3, 4, 5, 6, 7, or 8, c=2, 3, 4, 5, or 6;

[0169] In some embodiments, the structure of sp1 is specifically selected from the following group:

[0170] (1.1) a1=0, a2=2, b1=0, b2=0, b3=0, c=0;

[0171] (1.2) a1=0, a2=3, b1=0, b2=0, b3=0, c=0;

[0172] (1.3) a1=0, a2=4, b1=0, b2=0, b3=0, c=0;

[0173] (1.4) a1=0, a2=5, b1=0, b2=0, b3=0, c=0;

[0174] (1.5) a1=0, a2=6, b1=0, b2=0, b3=0, c=0;

[0175] (2.1) a1=0, a2=0, b1=0, b2=0, b3=0, c=2;

[0176] (2.2) a1=0, a2=0, b1=0, b2=0, b3=0, c=3;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0177] <h2 style=";text-align:left;direction:ltr"> (2.3)a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0178] <h2 style=";text-align:left;direction:ltr"> (2.4)a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0179] <h2 style=";text-align:left;direction:ltr"> (2.5)a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0180] <h2 style=";text-align:left;direction:ltr"> (3.1)a1 = 1, a2 = 2, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0181] <h2 style=";text-align:left;direction:ltr"> (3.2)a1 = 1, a2 = 2, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0182] <h2 style=";text-align:left;direction:ltr"> (3.3)a1 = 1, a2 = 2, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0183] <h2 style=";text-align:left;direction:ltr"> (3.4)a1 = 1, a2 = 2, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0184] <h2 style=";text-align:left;direction:ltr"> (3.5)a1 = 1, a2 = 2, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0185] <h2 style=";text-align:left;direction:ltr"> (3.6)a1 = 1, a2 = 2, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0186] <h2 style=";text-align:left;direction:ltr"> (3.7)a1 = 1, a2 = 2, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0187] <h2 style=";text-align:left;direction:ltr"> (3.8)a1 = 1, a2 = 3, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0188] <h2 style=";text-align:left;direction:ltr"> (3.9)a1 = 1, a2 = 3, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0189] <h2 style=";text-align:left;direction:ltr"> (3.10)a1 = 1, a2 = 3, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0190] <h2 style=";text-align:left;direction:ltr"> (3.11)a1 = 1, a2 = 3, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0191] <h2 style=";text-align:left;direction:ltr"> (3.12)a1 = 1, a2 = 3, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0192] <h2 style=";text-align:left;direction:ltr"> (3.13)a1 = 1, a2 = 3, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0193] <h2 style=";text-align:left;direction:ltr">(3.14)a1 = 1, a2 = 3, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0194] <h2 style=";text-align:left;direction:ltr"> (3.15)a1 = 1, a2 = 4, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0195] <h2 style=";text-align:left;direction:ltr"> (3.16)a1 = 1, a2 = 4, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0196] <h2 style=";text-align:left;direction:ltr"> (3.17)a1 = 1, a2 = 4, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0197] <h2 style=";text-align:left;direction:ltr"> (3.18)a1 = 1, a2 = 4, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0198] <h2 style=";text-align:left;direction:ltr"> (3.19)a1 = 1, a2 = 4, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0199] <h2 style=";text-align:left;direction:ltr"> (3.20)a1 = 1, a2 = 4, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0200] <h2 style=";text-align:left;direction:ltr"> (3.21)a1 = 1, a2 = 4, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0201] <h2 style=";text-align:left;direction:ltr"> (4.1)a1 = 0, a2 = 2, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0202] <h2 style=";text-align:left;direction:ltr"> (4.2)a1 = 0, a2 = 2, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0203] <h2 style=";text-align:left;direction:ltr"> (4.3)a1 = 0, a2 = 2, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0204] <h2 style=";text-align:left;direction:ltr"> (4.4)a1 = 0, a2 = 2, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0205] <h2 style=";text-align:left;direction:ltr"> (4.5)a1 = 0, a2 = 2, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0206] <h2 style=";text-align:left;direction:ltr"> (4.6)a1 = 0, a2 = 2, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0207] <h2 style=";text-align:left;direction:ltr"> (4.7)a1 = 0, a2 = 2, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0208] <h2 style=";text-align:left;direction:ltr"> (4.8)a1 = 0, a2 = 3, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0209] <h2 style=";text-align:left;direction:ltr">(4.9)a1 = 0, a2 = 3, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0210] <h2 style=";text-align:left;direction:ltr"> (4.10)a1 = 0, a2 = 3, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0211] <h2 style=";text-align:left;direction:ltr"> (4.11)a1 = 0, a2 = 3, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0212] <h2 style=";text-align:left;direction:ltr"> (4.12)a1 = 0, a2 = 3, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0213] <h2 style=";text-align:left;direction:ltr"> (4.13)a1 = 0, a2 = 3, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0214] <h2 style=";text-align:left;direction:ltr"> (4.14)a1 = 0, a2 = 3, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0215] <h2 style=";text-align:left;direction:ltr"> (4.15)a1 = 0, a2 = 4, b1 = 1, b2 = 2, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0216] <h2 style=";text-align:left;direction:ltr"> (4.16)a1 = 0, a2 = 4, b1 = 1, b2 = 3, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0217] <h2 style=";text-align:left;direction:ltr"> (4.17)a1 = 0, a2 = 4, b1 = 1, b2 = 4, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0218] <h2 style=";text-align:left;direction:ltr"> (4.18)a1 = 0, a2 = 4, b1 = 1, b2 = 5, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0219] <h2 style=";text-align:left;direction:ltr"> (4.19)a1 = 0, a2 = 4, b1 = 1, b2 = 6, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0220] <h2 style=";text-align:left;direction:ltr"> (4.20)a1 = 0, a2 = 4, b1 = 1, b2 = 7, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0221] <h2 style=";text-align:left;direction:ltr"> (4.21)a1 = 0, a2 = 4, b1 = 1, b2 = 8, b3 = 0, c = 0<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0222] <h2 style=";text-align:left;direction:ltr"> (5.1)a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, c = 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0223] <h2 style=";text-align:left;direction:ltr"> (5.2)a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 3, c = 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0224] <h2 style=";text-align:left;direction:ltr"> (5.3)a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 4, c = 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0225] (5.4) a1=1, a2=0, b1=0, b2=0, b3=5, c=2;

[0226] (5.5) a1=1, a2=0, b1=0, b2=0, b3=6, c=2;

[0227] (5.6) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 7, c = 2; and

[0228] (5.7) a1=1, a2=0, b1=0, b2=0, b3=8, c=2.

[0229] In some embodiments, the cleavable linker L2 is a dipeptide, tripeptide, or tetrapeptide residue.

[0230] In some embodiments, L2 is selected from the following dipeptide residues: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Val-Cit-, -Ala-Lys-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, -Gly-Gly-, -Ala-Ala-, -Gly-Val-, and -Gly-Glu-; the left side of the dipeptide residue is connected to sp1 and the right side is connected to sp2. Preferably, L2 is selected from -Val-Ala-, -Val-Lys-, and -Val-Cit-.

[0231] In some embodiments, L2 is a tripeptide residue selected from the group consisting of: -Glu-Val-Ala-, -Glu-Val-Cit-, -αGlu-Val-Ala-, -αGlu-Val-Cit-, -Val-Lys-Gly, and -Val-Cit-Gly-; the left side of the tripeptide residue is linked to sp1 and the right side is linked to sp2.

[0232] In some embodiments, L2 is a tetrapeptide residue selected from the group consisting of: -Gly-Gly-Phe-Gly- and -Gly-Phe-Gly-Gly-; the left side of the tetrapeptide residue is linked to sp1 and the right side is linked to sp2.

[0233] In some embodiments, the second spacer unit sp2 is absent, or sp2 is selected from:

[0234] (The side connected to L2 is marked as L2, and the side connected to the biologically active substance D is marked as D), wherein,

[0235] R2 is independently selected from hydrogen, C 1-6 Alkyl, hydroxy, amino, halogen, nitro, cyano, d is an integer from 1 to 20, e is an integer from 1 to 20; R3 and R4 are each independently selected from hydrogen and C 1-6 alkyl;

[0236] The alkyl group may be optionally substituted with hydroxy, amino, halogen, nitro and cyano.

[0237] In some embodiments, sp2 is

[0238] In some embodiments, sp2 is

[0239] In some embodiments, sp2 is selected from: wherein d is an integer from 1 to 10, and e is an integer from 1 to 10.

[0240] In some embodiments, the -L1-sp1-L2-sp2- is selected from the following structures:

[0241] (the side connected to the protein P is marked as P, and the side connected to the biologically active substance D is marked as D); wherein k is an integer from 1 to 20.

[0242] In some embodiments, k is an integer from 2 to 16, for example, k=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0243] In some embodiments, the -L1-sp1-L2-sp2- is further selected from the following structures:

[0244] Wherein, the definition of k is as described above.

[0245] In some embodiments, the biologically active substance D can be selected from cytotoxins, protein kinase inhibitors, immune agonists, glucocorticoids, oligonucleotides, radioisotopes, polypeptides, and any combination thereof.

[0246] In some embodiments, D is a cytotoxin selected from the group consisting of: a DNA alkylating agent, a DNA destructuring agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule inhibitor, a ribosome inhibitor, and any combination thereof.

[0247] In some specific embodiments, D is selected from: Auristatin derivatives, Maitansine derivatives, Eribulin derivatives, tubulysin derivatives, Pyrrolobenzodiazepine (PDB) derivatives, Duocarmycin derivatives, Calicheoamicin derivatives, PNU-159682 and its derivatives, Camptothecin derivatives, Amatoxin derivatives and any combination thereof.

[0248] In some embodiments, D is selected from camptothecin derivatives, which refer to compounds having the same 5-membered syntenic core structure as naturally derived camptothecin and having substitution modifications at positions 7, 9, 10, and 11, and which have the same or stronger topoisomerase I inhibitory activity as naturally derived camptothecin.

[0249] Optional camptothecin derivatives can be derived from the prior art as a whole, such as patent application documents WO2014057687, WO2020063676, CN111689980A, WO2022068878, WO2022068878, WO2020259258, WO2020219287, WO2022121981, WO2021173773, WO2019195665, WO2021067861, WO2022170971, WO2020200880, WO2021148501, WO2023109965 and WO2022015656, the disclosures of the above patents are incorporated into the present application as a whole.

[0250] In some embodiments, D has the structure shown in Formula III:

[0251] wherein X is selected from CH2, NH, O, S or SO2;

[0252] Y does not exist, or Y has The structure shown;

[0253] wherein W1 and W3 are each independently selected from O, S and NH, and W2 is selected from C, CH and N,

[0254] R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano and nitro; or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl or a carbonyl group; or, R a is connected to the N atom of the amide portion to form a 3-6 membered heterocycloalkyl group and R b is hydrogen;

[0255] Ring A is selected from the group consisting of a 5-10 membered cycloalkylene, a 5-10 membered heterocycloalkylene, a 6-10 membered arylene, and a 5-10 membered heteroarylene;

[0256] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano, carbonyl and nitro;

[0257] d and e are each independently selected from integers from 0 to 5.

[0258] In some embodiments, D has the structure shown in Formula III-a:

[0259] Where W1 is O or NH;

[0260] R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano and nitro; or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl or a carbonyl group; or, R a is connected to the N atom of the amide portion to form a 3-6 membered heterocycloalkyl group and R b is hydrogen;

[0261] The alkyl, alkoxy, cycloalkyl and heterocycloalkyl groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano and nitro;

[0262] d is an integer from 0 to 5.

[0263] In some embodiments, W1 is O.

[0264] In some embodiments, d is 0, 1, or 2.

[0265] In some embodiments, wherein R a Selected from hydrogen, deuterium, halogen, C1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxyl and amino, R b The alkyl, alkoxy, cycloalkyl and heterocycloalkyl groups are each independently optionally further substituted by a group selected from deuterium, halogen, hydroxy, amino, cyano and nitro.

[0266] In some embodiments, wherein R a and R b Together with the carbon atoms to which they are attached, they form a 3-6 membered cycloalkyl or a 3-6 membered heterocycloalkyl. The cycloalkyl and heterocycloalkyl are each independently optionally further substituted by a group selected from deuterium, halogen, hydroxy, amino, cyano and nitro.

[0267] In some embodiments, D can be selected from the group consisting of:

[0268] (i.e. DXd),

[0269] In other embodiments, D has the structure shown in Formula III-b:

[0270] Where W3 is O or NH, W2 is C, CH or N,

[0271] Ring A is selected from the group consisting of a 5-10 membered cycloalkylene, a 5-10 membered heterocycloalkylene, a 6-10 membered arylene, and a 5-10 membered heteroarylene;

[0272] The cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl and nitro;

[0273] e is an integer from 0 to 5.

[0274] In some embodiments, W3 is O; in other embodiments, W2 is CH.

[0275] In some embodiments, e is 0, 1, or 2.

[0276] In some embodiments, Ring A is a 5-10 membered cycloalkylene group. The cycloalkylene group may be further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl, and nitro.

[0277] In some embodiments, D can be selected from the group consisting of:

[0278] In some embodiments, D, as a camptothecin derivative, can also be selected from the following structures:

[0279] (i.e. Exatecan), (i.e. SN-38).

[0280] In some specific embodiments, the -L1-sp1-L2-sp2-D is selected from the following structures:

[0281] Here, k is an integer from 1 to 20.

[0282] In some embodiments, k is an integer from 2 to 16, for example, k=2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0283] In some embodiments, n=1-15, eg, n=1-10, n=1-8, n=2-6, n=2-5, or n=2.5-3.5.

[0284] In some embodiments, in Formula I, the linker L and the protein P are connected via a thiol group; preferably, the thiol group is derived from an antibody; more preferably, the thiol group is obtained by reducing the disulfide bonds between heavy chains and / or the disulfide bonds between heavy chains and light chains.

[0285] In other embodiments, the linker L is linked to the protein P via an oligosaccharide; preferably, the oligosaccharide is derived from the natural sugar chain of an antibody.

[0286] In some embodiments, the oligosaccharide is derived from an N-glycan chain of an antibody.

[0287] In some embodiments, the oligosaccharide consists of 2 to 15 monosaccharides; preferably, the oligosaccharide consists of 2 to 10 monosaccharides.

[0288] In some embodiments, the oligosaccharide has the structure shown in Formula Va or Formula Vb:

[0289] wherein P* binds to HER3 and TROP2, for example, it comprises the antigen binding protein described herein, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;

[0290] Gal* is a modified galactose selected from the following structures:

[0291] The oligosaccharide is linked to P* via a core GlcNAc.

[0292] In some embodiments, the modified galactose is linked to the GlcNAc via a β-1,4-glycosidic bond.

[0293] In some embodiments, the oligosaccharide is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to Asn297 (numbering according to EU index of Kabat) of the Fc fragment.

[0294] In some specific embodiments, the protein-drug conjugate described herein has a structure shown in Formula VI:

[0295] wherein P* binds to HER3 and TROP2, for example, it comprises the antigen binding protein described herein, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;

[0296] Gal* is a modified galactose selected from the following structures:

[0297] LP is selected from one of the following structures (a) to (f):

[0298] (a) and / or

[0299] (b) and / or

[0300] (c) and / or as well as

[0301] (d) and / or k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0302] Among them, the core GlcNAc is directly connected to P*.

[0303] In some embodiments, the DAR value of the conjugate as a whole is from 1 to 8.

[0304] In some embodiments, j=1-10, for example, j=1-8, j=1.2-6, j=1.5-3, j=1.5-2.5.

[0305] In some embodiments, the antigen binding protein comprises a first polypeptide represented by SEQ ID NO:51 and a second polypeptide represented by SEQ ID NO:50.

[0306] In some embodiments, in the protein-drug conjugate, the oligosaccharide is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to Asn297 (numbering according to the EU index of Kabat) of the Fc fragment.

[0307] As mentioned above, the oligosaccharide can be derived from the natural sugar chain of the antibody. Specifically, the method for preparing the oligosaccharide precursor (or referred to as a protein derivative herein) comprises the following steps: reacting an antibody whose N-sugar glycoform is mainly G0F / G0 with UDP-GalNAz or a salt thereof, or with other UDP-GalNAc azide derivatives, in the presence of a catalyst to obtain the aforementioned oligosaccharide precursor.

[0308] The UDP-GalNAz has the following structure:

[0309] Methods for obtaining antibodies with G0F / G0 glycoforms are well known in the art. For example, antibodies expressed in eukaryotic cells are post-translationally modified and the glycans can be converted to the G0F / G0 form by treatment with β-galactosidase, which removes any terminal galactose residues and leaves a terminal N-acetylglucosamine residue.

[0310] In other embodiments, the starting sugar chain of the G0F / G0 glycoform described in the present disclosure can also be obtained by expressing and purifying the B4GALT1 gene in a cell line in which the B4GALT1 gene is knocked out. One example of knocking out the B4GALT1 gene in a cell line expressing the B4GALT1 gene is through homologous recombination technology. Other examples of knocking out the B4GALT1 gene include using zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs). The specific methods are described in reports such as Nature Biotechnology, volume 33, pages 842-844 (2015). Int. J. Mol. Sci. 2015, 16 (10), 23849-23866.

[0311] In some embodiments, the aforementioned catalyst is galactosyltransferase or a functional variant or fragment thereof.

[0312] In some embodiments, the catalyst is β-1,4-galactosyltransferase or a functional variant or fragment thereof.

[0313] In some embodiments, the catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase, or a functional variant or fragment thereof.

[0314] In some embodiments, the catalyst is human β-(1,4)-GalT1 with a Y285L mutation or bovine β-(1,4)-GalT1 with a Y289L mutation.

[0315] In some embodiments, the catalyst is β-1,4-acetylgalactosyltransferase disclosed in patent application WO2016170186.

[0316] In some embodiments, the catalyst comprises the sequence set forth in any one of SEQ ID NOs: 52-54.

[0317] Protein derivatives

[0318] On the other hand, the present application also relates to a protein derivative having a structure shown in Formula VII:

[0319] wherein P* binds to HER3 and TROP2, comprises the antigen binding protein described herein, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20;

[0320] Gal** is a modified galactose selected from the following structures:

[0321] The oligosaccharide is linked to P* via a core GlcNAc.

[0322] The protein derivative can be obtained by reacting an antigen-binding protein whose N-sugar glycoform is mainly G0F with UDP-GalNAz or its salt, or with other UDP-GalNAc azide derivatives in the presence of a catalyst.

[0323] Nucleic acid of the present invention and vector and host cell containing the same

[0324] In one aspect, the invention provides nucleic acids encoding any of the above binding proteins, single variable domains, VHHs, fusion proteins, antibodies, multispecific antigen-binding proteins, or any fragment thereof (e.g., antigen / target binding fragments). The invention also encompasses nucleic acids that hybridize to the above nucleic acids under stringent conditions, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions compared to the above nucleic acids, or nucleic acid sequences that are at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the above nucleic acids.

[0325] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-51, or nucleic acids encoding 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 any one of SEQ ID NOs: 1-51.

[0326] As will be appreciated by those skilled in the art, due to codon degeneracy, the amino acid sequence of each binding protein, single variable domain, VHH, fusion protein, antibody, multispecific antigen-binding protein, or any fragment thereof, can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding 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.

[0327] In one aspect, the present invention provides nucleic acids encoding any of the above binding proteins, single variable domains, VHHs, fusion proteins, antibodies, multispecific antigen-binding proteins, or any peptide chains / fragments thereof. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human HER3 and / or TROP2 binding ability.

[0328] In one embodiment, the nucleic acids encoding each chain of a binding protein, single variable domain, VHH, fusion protein, antibody, or multispecific antigen-binding protein of the present invention can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of a binding protein, single variable domain, VHH, fusion protein, antibody, or multispecific antigen-binding protein of the present invention can be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing a binding protein, single variable domain, VHH, fusion protein, antibody, or multispecific antigen-binding protein of the present invention comprises the steps of culturing a host cell containing nucleic acids encoding each chain under conditions suitable for expression of each chain of the molecule, thereby producing an antibody, bispecific binding molecule, fusion protein, or fragment thereof, or multispecific binding molecule of the present invention.

[0329] On the other hand, the present invention provides a vector comprising the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It is fully understood by those skilled in the art that the vectors commonly used in the technical field to which the present invention belongs can be applied to the present invention.

[0330] In one embodiment, the present invention provides a host cell comprising the nucleic acid or the vector.

[0331] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity that have been screened or selected in the initially transformed cells. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0332] Composition

[0333] In another aspect, the present disclosure provides a composition, e.g., preferably, a pharmaceutical composition, containing one or a combination of HER3 binding proteins, fusion proteins, multispecific antigen-binding proteins, or protein-drug conjugates of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0334] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0335] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition that produces a therapeutic effect. Typically, based on 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, for example, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0336] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure being administered, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of treatment, other drugs, compounds, and / or materials being administered in combination with the particular composition being administered, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0337] In some embodiments, the DAR value of the disclosed protein-drug conjugate compositions is from about 1.0 to 16.0, preferably from about 2.0 to 12.0, more preferably from about 3.0 to 6.0, and even more preferably from about 3.5 to 4.5. For example, the DAR value of the protein-drug conjugate composition is about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0.

[0338] The compositions of the present disclosure can be administered by one or more methods well known in the art through one or more routes of administration. It will be understood by those skilled in the art that the route of administration and / or mode are different according to the desired result. The preferred route of administration of the HER3 binding proteins, multispecific antigen binding proteins or protein-drug conjugates of the present disclosure includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to the mode of administration other than enteral and topical administration, typically injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0339] Drug combinations

[0340] 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. The term "non-fixed combination" means that the active ingredients (e.g., (i) the HER3 binding molecules of the present invention, (ii) the multispecific antigen binding proteins of the present invention, and (iii) the protein-drug conjugates of the present invention) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with a preventive or therapeutically effective level in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts 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 the form of a separate formulation, which can be the same or different.

[0341] Therefore, in another aspect, the present application also provides a pharmaceutical combination or combination product comprising the binding protein, single variable domain, VHH, fusion protein, antibody, multispecific antigen-binding protein, and protein-drug conjugate of the present invention, wherein the definitions of the binding protein, single variable domain, VHH, fusion protein, antibody, multispecific antigen-binding protein, and protein-drug conjugate in the combination can refer to the technical features described in the first aspect. In some embodiments, the pharmaceutical combination or combination product may further comprise one or more other therapeutic agents, such as chemotherapeutic agents.

[0342] The present application also provides a kit comprising the drug combination, for example, the kit comprises in the same package:

[0343] - a first container containing a binding protein, single variable domain, VHH, fusion protein, antibody, multispecific antigen-binding protein, nucleic acid, vector, host cell of the present invention, or a pharmaceutical composition comprising the same;

[0344] - a second container containing the protein-drug conjugate of the present invention or a pharmaceutical composition comprising the protein-drug conjugate;

[0345] The definitions of binding proteins, single variable domains, VHHs, fusion proteins, antibodies, multispecific antigen-binding proteins, nucleic acids, vectors, host cells, and protein-drug conjugates can refer to the technical features described in the aforementioned aspects of the present invention.

[0346] In some embodiments, the kit further comprises, in the same package, an additional container containing or comprising an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0347] Treatment methods and uses

[0348] In another aspect, the present application also provides a method for treating a tumor or cancer, such as a solid tumor or non-solid tumor, such as a hematological tumor, comprising administering to a patient in need thereof one of the binding proteins, single variable domains, VHHs, fusion proteins, antibodies, multispecific antigen-binding proteins, nucleic acids, vectors, host cells, and protein-drug conjugates of the present invention, or a combination / combination of more than one thereof. The present application also provides a method for treating a tumor or cancer, such as an advanced or metastatic solid malignant tumor, comprising administering to a patient in need thereof a pharmaceutical composition of the present application.

[0349] In this application, the phrases "administering a drug combination..." and "...in combination..." encompass both simultaneous administration of multiple drugs and sequential administration of multiple drugs. When administered sequentially, the interval between administration of the multiple drugs is no more than 24 hours, for example, no more than 18 hours, no more than 15 hours, no more than 12 hours, no more than 10 hours, no more than 8 hours, no more than 5 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, or no more than 0.5 hours.

[0350] On the other hand, the present application also relates to the use of the HER3 binding protein, multispecific antigen binding protein, nucleic acid, vector, host cell and / or protein-drug conjugate in the preparation of a drug, which can be used to treat and / or prevent tumors.

[0351] In some embodiments, the tumor comprises a solid tumor and / or a non-solid tumor, such as a hematological tumor. In some embodiments, the tumor is HER3 positive and / or TROP2 positive. In some embodiments, the tumor is a tumor whose treatment would benefit from inhibition of HER3 and / or TROP2.

[0352] Example

[0353] Instruments and Equipment

[0354] Reagents and consumables

[0355] Example 1 Screening, Preparation and Characterization of HER3 Single Domain Antibodies

[0356] 1.1 Construction of HER3 single-domain antibody immune library

[0357] Healthy camels were selected and injected with human HER3-Fc fusion protein as an antigen at multiple points in the neck muscle. Immunizations were repeated every two weeks for a total of six times. At the end of the final immunization, 50 mL of peripheral blood was collected into a vacuum tube, and the supernatant was collected as post-immune serum. Lymphocytes were isolated by density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was completely reverse transcribed into cDNA using the Super-Script III FIRST STRAND SUPERMIX kit (ThermoFisher) according to the manufacturer's instructions. Nucleic acid fragments encoding the variable regions of the heavy chain antibodies were amplified by nested PCR.

[0358] The nucleic acid fragment of the target heavy chain single domain antibody was recovered and cloned into the phage display vector pComb3XSS using the restriction endonuclease SfiI. The product was then electroporated into E. coli electrocompetent cells TG1 to construct and characterize an anti-HER3 immune single domain antibody phage display library. The library size was calculated to be approximately 1.0×10 8 .

[0359] 1.2 Selection of HER3 single-domain antibodies

[0360] The phage library obtained above was panned. In the first round of panning, HER3-muFc (human HER3-mouse Fc fusion protein) was used as the screening source and PD-L1-Fc as the negative screening source. In the second and third rounds of panning, HER3-muFc was used as the screening source and SP-Fc (signal peptide-Fc fusion protein) was used as the negative screening source. Clones that bound to HER3-muFc but not to Fc were obtained by panning.

[0361] The binding-positive phages obtained after the above panning were infected with blank E. coli and plated. Subsequently, colonies were selected and inoculated into 2TY-AG (containing 10% glycerol) and allowed to stand at room temperature overnight. The next day, 1% of the inoculum was transferred to 200 μL of 2TY-AG and cultured at 37°C with shaking at 250 rpm until the OD600 reached approximately 0.5. Helper phage M13KO7 was added for infection (infection index of 1:20), and the culture was allowed to stand at 37°C for 15 minutes, followed by 220 rpm for 45 minutes. 800 μL of 2TY-AG was added to each well and the culture was incubated at 30°C, 220 rpm, overnight. The next day, the supernatant was collected for ELISA detection. Plates were coated with HER3-Fc and SP-Fc respectively overnight at 4°C, and the obtained supernatant was added and reacted at room temperature for 2 hours. After washing, the secondary antibody, Goat anti-HA tag HRP (Abeam) or Goat anti-mouse IgG-HRP (Thermo), was added and incubated at room temperature for 2 hours. After washing, TMB colorimetric solution was added and the absorbance at 450 nm and 650 nm was read. The final absorbance was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm. The results are shown in Table 1.

[0362] Table 1 OD values ​​of positive clones

[0363] The positive clones that specifically bind to HER3 were sequenced, and the amino acid sequence of iBT11 is shown below:

[0364] 1.3 Preparation of HER3 single-domain antibodies and their Fc fusion proteins

[0365] PCR amplify the HER3 single-domain antibody VHH coding sequence fragment, fuse it with a DNA fragment encoding human IgG1-Fc, or fuse it with a His tag (Chis) at its C-terminus, and clone it into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the HER3 single-domain antibody-Fc / Chis fusion protein in mammals. The universal primers used for PCR amplification are as follows:

[0366] Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 57)

[0367] Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO: 58)

[0368] The constructed plasmid vector was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle 293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was added to the HEK293 cell suspension and placed at 37°C, 5% CO2 for suspension culture. After 5-6 days of culture, the supernatant of the transient expression culture was collected and purified by Protein A affinity chromatography or Ni column to obtain the target HER3 single-domain antibody-Fc / Chis fusion protein.

[0369] 1.4 Detection of affinity for HER3

[0370] The affinity of the HER3 single-domain antibody was tested using ELISA. 5 μg / mL of Her3-muFc was coated and incubated overnight at 4°C. The HER3 single-domain antibody, Chis, was serially diluted in 1% BSA + 0.05% PBST20, starting at a concentration of 5 μg / mL. The dilutions were repeated three-fold, with a total of 10 dilutions, and the reaction was allowed to proceed at room temperature for 2 hours. After washing with 1% BSA + 0.05% PBST20, the secondary antibody, anti-6*his tag HRP (purchased from abcam, 1:10,000 dilution), was added. After washing with 1% BSA + 0.05% PBST20, TMB colorimetric solution was added and the plate was developed at room temperature for 5 minutes. The absorbance at 450 nm and 650 nm was read. The final absorbance was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm. The results are shown in Figure 1. The EC50 value of iBT11-Chis was 5.39 ng / mL.

[0371] The binding kinetics of HER3 single-domain antibody-Fc fusion protein to HER3-chis were detected by biolayer interferometry (BLI) technology. The iBT11-Fc fusion protein was diluted to 10 ug / mL and then immobilized on the AHC biosensor. HER3-chis was then diluted to five gradients of 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM, and then bound to the immobilized iBT11-Fc fusion protein. The equilibrium dissociation constant (KD) was calculated using Octet K2 data analysis software 9.0, and the results are shown in Table 2.

[0372] Table 2 Affinity results of iBT11-Fc (BLI)

[0373] 1.5 Humanization of HER3 single-domain antibodies

[0374] The humanization method uses the protein surface amino acid humanization (resurfacing) method and VHH humanization universal framework transplantation method (CDR grafting to a universal framework) to complete.

[0375] First, we obtained the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK), designed by Cécile Vincke et al. based on sequence homology. This framework is based on the nanobody NbBcII10 (PDB ID: 3DWT). We performed modeling using Modeller9, and calculated the relative solvent accessibility of the amino acids on the framework based on the protein's three-dimensional structure.

[0376] The specific steps for the universal framework transplantation method for VHH humanization are as follows: A highly homologous human antibody sequence is obtained through IMGT. The target sequence is then humanized using the universal humanized VHH framework hNbBcII10FGLA (PDB ID: 3EAK) as a reference. Using the highly homologous framework as a framework template, the CDRs are replaced with the CDR regions of the target antibody strain. Based on the modeling, non-surface amino acids on the framework are then backmutated to complete the humanization of the target antibody.

[0377] The antibody strain iBT11 was humanized to obtain humanized variants huiBT11v1 (SEQ ID NO: 2), huiBT11v3 (SEQ ID NO: 3), huiBT11v4 (SEQ ID NO: 4), huiBT11v5 (SEQ ID NO: 5), huiBT11v6 (SEQ ID NO: 6), and huiBT11v7 (SEQ ID NO: 7).

[0378] The humanized sequence was synthesized, fused with a DNA fragment encoding human IgG1-Fc, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the HER3 single-domain antibody-Fc fusion protein in mammals. The constructed vector was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle 293 medium and the PEI (Polyethylenimine) solution required for transformation was added. Each plasmid / PEI mixture was added to the HEK293 cell suspension and incubated at 37°C, 5% CO2, and 130 rpm. After four hours, EXCELL 293 medium with 2mM glutamine was added and incubated at 130 rpm. After 24 hours, 3.8mM VPA was added, and after 72 hours, 4g / L glucose was added. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target huiBT11 single-domain antibody Fc fusion protein.

[0379] 1.6 Testing the affinity of humanized antibodies

[0380] The affinity of the humanized antibody for HER3 was assessed by ELISA. The plates were coated with 5 μg / mL of Her3-muFc at 4°C overnight. The antibody was serially diluted in 1% BSA + 0.05% PBST20, starting at 100 μg / mL. Four-fold dilutions were added, totaling 10 steps, and the plates were incubated at room temperature for 2 hours. After washing with 1% BSA + 0.05% PBST20, the secondary detection antibody, Goat anti-human IgG (Fc specific)-HRP (Sigma, 1:8000 dilution), was added and incubated at room temperature for 2 hours. After washing with 1% BSA + 0.05% PBST20, TMB colorimetric solution was added and the plate was developed at room temperature for 5 minutes. The absorbance at 450 nm and 650 nm was read. The final absorbance was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm. The results are shown in Table 3.

[0381] The sequence of the positive control antibody AV203 is referenced from patent document WO2011136911: AV203HC (SEQ ID NO: 55) and AV203LC (SEQ ID NO: 56) were synthesized and cloned into conventional mammalian expression vectors, and the constructed vectors were transfected into HEK293 cells for transient expression of the antibodies.

[0382] Table 3 Affinity results of humanized antibodies (ELISA method)

[0383] 1.7 Detection of blocking activity of humanized antibodies

[0384] ELISA was used to detect the blocking activity of humanized antibodies against HER3-muFc and NRG1b1 binding. 1 μg / mL of NRG1 beta1 (purchased from Sino) and SP-NRG1b-Chis were coated overnight at 4°C. The test antibody was serially diluted with 1% BSA + 0.05% PBST20 + 200 ng / mL Her3-muFc, starting at 20 μg / mL. Six 4-fold dilutions were performed and the plates were incubated at room temperature for 2 hours. After washing with 1% BSA + 0.05% PBST20, the secondary detection antibody, Goat anti-mouse IgG1-HRP (purchased from Thermo, diluted 1:3000), was added and incubated at room temperature for 2 hours. After washing with 1% BSA + 0.05% PBST20, TMB colorimetric solution was added and color was developed at room temperature for 7.5 minutes. The absorbance at 450 nm and 650 nm was read. The final absorbance was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm. The results are shown in Table 4.

[0385] Table 4 Blocking activity results of humanized antibodies (ELISA method)

[0386] Example 2 Preparation of Antibody-Drug Conjugates

[0387] 2.1 Preparation of Antibody A

[0388] Genes were synthesized based on the sequences of the first polypeptide (SEQ ID NO: 51) and the second polypeptide (SEQ ID NO: 50) of the target bispecific antibody (Antibody A), amplified by PCR using primers, and cloned into the conventional mammalian expression vector pcDNA3.4 to obtain the pcDNA3.4-A recombinant plasmid.

[0389] HEK293 cells were cultured in 293M medium at 37°C, 130 ± 10 rpm, and 5% CO2. Before transient transfection, the density of HEK293 cells was adjusted to 6 × 10 6 cells / mL. Mix the pcDNA3.4-A plasmid and the transfection reagent PEI in a 1:6 ratio. Add the mixed transfection reagent and plasmid complex to approximately 2 mL of OPM-293-CD05 expression medium and let it stand for 5 minutes. Add the above solution to 100 mL of expression medium and incubate at 37°C, 130±10 rpm, 5% CO2. On day 7, centrifuge at 1500 rpm for 10 minutes and harvest the supernatant. Purify the supernatant by Protein A affinity purification to obtain the target protein, and verify the correct size of the target protein by SDS PAGE.

[0390] 2.2 Preparation of Linker-Toxin

[0391] The following linker-toxin (LP1) was prepared according to the process described in patent application CN113264983A (the contents of which are incorporated herein by reference):

[0392] After detection, LP1 MS m / z (ESI): 1375.77.

[0393] 2.3 Preparation of Antibody A-ADC

[0394] Step 1: Desalt the stock solution of Antibody A (primarily G0F) into HEPES buffer for later use. To a 1.5 mL centrifuge tube, add the following: ultrapure water (210 μL), MnCl2 (1 M, 5 μL), UDP-GalNAz (100 mM, 25 μL), Tris-HCl (1 M, 5 μL), Antibody A (25.5 mg / mL, 196 μL), and GalT1 (5.2 mg / mL, 12 μL, SEQ ID NO: 52). Incubate at 30°C, 400 rpm for 12 h. After completion of the reaction, purify Protein A and concentrate by ultrafiltration into DPBS to obtain Antibody A-(N3)4 for later use.

[0395] Step 2: To a 1.5 mL centrifuge tube, add LP1 (16 μL, 50 mM), DMSO (25 μL), and antibody A-(N3)4 (10 mg / ml, 800 μL) obtained in step 1. Incubate at 30°C, 400 rpm for 12 h. After completion, desalt the mixture into DPBS to obtain antibody A-ADC. Mass spectrometry analysis of the molecular weight is shown in Figure 2. Compared to the unconjugated antibody A, the target product was confirmed. RP-HPLC analysis of the product revealed a DAR value of approximately 3.8. The overall reaction equation is shown in Figure 3.

[0396] Example 3 Characterization of the biological activity of antibody A-ADC

[0397] 3.1 Detection of Antibody A-ADC Binding Activity to TROP2 (BLI Method)

[0398] The affinity of Antibody A-ADC for human and monkey TROP2 was tested using Bio-Layer Interferometry (BLI) technology. Antibody A-ADC was first diluted to 10 μg / mL onto a proA biosensor immobilized with the protein. The TROP2 protein was then diluted to seven concentrations, each of which was bound to the Antibody A-ADC. Binding signals of varying intensities were detected. Data Analysis HT 12.0 software was used to analyze the data, and a 1:1 model was fitted to calculate the equilibrium dissociation constant (KD) for the samples. The results are shown in Table 5. The binding ability of Antibody A-ADC to human and monkey TROP2 proteins was comparable to that of the naked antibody.

[0399] Table 5 Affinity results of samples with human and monkey TROP2 proteins

[0400] 3.2 Detection of Antibody A-ADC Binding Activity to HER3 (BLI Method)

[0401] A similar method was used to test the affinity of Antibody A-ADC for human and monkey HER3 proteins. The results are shown in Table 6. Antibody A-ADC's binding ability to human and monkey HER3 proteins was comparable to that of the naked antibody.

[0402] Table 6 Affinity results of samples with human and monkey HER3 proteins

[0403] 3.3 Detection of the activity of antibody A-ADC binding to HER3 and TROP2 simultaneously

[0404] The ELISA method was used to detect the ability of antibody A-ADC to bind to HER3 and TROP2 proteins simultaneously. The test samples with multiple concentration gradients were added to the enzyme-labeled plate pre-coated with antigen 1 (HER3-His) and incubated, and then biotinylated antigen 2 (TROP2-Biotin, homemade) was added for incubation, and finally the detection antibody labeled with horseradish peroxidase (SA-HRP) was added to form a solid phase antigen 1-antibody A-ADC sample-antigen 2-enzyme-labeled detection antibody complex. The absorbance value was read at 450nm using an enzyme reader, and the data was analyzed by four-parameter fitting curve analysis. The results are shown in Table 7. Antibody A-ADC and antibody A can both bind to human HER3 and TROP2 proteins simultaneously, and bind to EC 50 The binding capacities of the two proteins were 31.42nM and 29.95nM respectively; iBT11V7-Fc and hRS7-IgG1 could not bind to two proteins at the same time.

[0405] Table 7 Binding activity results of Antibody A-ADC and Antibody A to human HER3 and TROP2 proteins

[0406] 3.4 Detection of Antibody A-ADC Binding Activity to Tumor Cells

[0407] 1.0×10 5 The human triple-negative breast cancer cell line MDA-MB-468, the human gastric cancer cell line NCI-N87, and the human pancreatic cancer cell line BxPC-3 (all purchased from Beina Chuanglian Biotechnology Co., Ltd.) were incubated with antibody A-ADC and antibody A (the highest final concentration was 200 nM, 3-fold serial dilution, a total of 10 concentrations) on ice for 30 min, washed twice, and then APC anti-human IgG Fc fluorescent antibody was added. The cells were incubated on ice in the dark for 30 min and washed twice. The cell surface fluorescence intensity was detected by flow cytometry. The flow cytometry results were analyzed using CytExpert software, the mean fluorescence intensity (MFI) of each test sample was calculated, and the experimental results were fitted with four parameters using GraphPad Prism 7.0.

[0408] The results are shown in Table 8. Antibody A-ADC and naked anti-antibody A have similar binding activities to tumor cells.

[0409] Table 8 Binding activity of antibody A-ADC to tumor cells

[0410] 3.5 Detection of the bridging effect of antibody A-ADC on 293T-TROP2 and 293T-HER3 cells

[0411] 293T-TROP2-GFP cells (1.0×10 5 / well, HEK293T cells expressing TROP2 on the cell surface and green fluorescent protein in the cells, homemade) and 293T-HER3-CellTrace Violet cells (1.0×10 5 / well, cells expressing HER3 on their surface and tested by CellTrace TMViolet fluorescent dye-labeled cells (purchased from Kangyuan Bochuang Biotechnology Co., Ltd.) were mixed in a 1:1 ratio with a mixture of Antibody A-ADC, Antibody A, iBT11V7-FC, and hRS7-IgG1 at concentrations ranging from 0.016 to 50 nM. The cells were then incubated on ice for 30 minutes in the dark. After washing twice, the fluorescence signals of 293T-HER3-CellTrace Violet cells and 293T-TROP2-GFP were collected in the PB450 / FITC channels of a CytoFelx flow cytometer. The dual fluorescence signal events in the 2D scatter plots of the PB450 and FITC channels demonstrate the bridging effect of Antibody A-ADC on the two target cells.

[0412] The results, as shown in Table 9, show that with increasing concentrations of Antibody A-ADC, the percentage of dual fluorescence signal events in the test samples increased from 1.82% to 22.46%, demonstrating a clear concentration-dependence. However, with further increases in drug concentration, the percentage of dual fluorescence signal events decreased, impairing the ability of Antibody A-ADC to bridge the two cell types, resulting in a "hook effect." Compared to Antibody A-ADC, Antibody A demonstrated comparable bridging capabilities. These results demonstrate that Antibody A, as an anti-HER3 and TROP2 bispecific antibody-drug conjugate, can simultaneously bind to both TROP2- and HER3-positive cells, demonstrating the ability to bridge the two cell types. In contrast, a mixture of an anti-TROP2 monoclonal antibody and an anti-HER3 single-domain antibody Fc fusion protein showed no significant bridging effect.

[0413] Table 9 Bridging effect of antibody A-ADC on TROP2 and HER3 positive cells

[0414] 3.6 Detection of Antibody A-ADC’s ​​Killing Effect on Tumor Cells

[0415] Human pancreatic cancer BxPC-3 cells were cultured at 5×10 3 / well plated 96-well plate, after overnight culture, add antibody A-ADC, antibody A and iso-ADC (isotype control ADC drug, the antibody is human IgG1 isotype control, the toxin linker is the same as antibody A-ADC, obtained by the same preparation process as antibody A-ADC) at a final concentration of 0.006-25nM, cultured at 37 ° C for 120h, and the OD value at 450 / 650nm of the microplate reader was detected using the CCK-8 method. The cell viability of each experiment was calculated, and the inhibition rate of each experimental group was calculated. The logarithm value (x) of the standard curve sample concentration and the calculated inhibition rate (%) (y) were then fitted using the 4-parameter logistic fit of GraphPad Prism7.0 software to calculate the IC of the sample to be tested. 50The experimental results are shown in Figure 4. Antibody A-ADC has a significant killing effect on BxPC-3 cells. 50 was 0.141nM; while antibody A and iso-ADC had no obvious killing effect on the proliferation of BxPC-3 cells.

[0416] A similar method was used to test the killing effect of antibody A-ADC on human epidermal cancer cell line A431 (purchased from Beina Chuanglian Biotechnology Co., Ltd., final sample concentration 0.003-50 nM), human pancreatic cancer cell line CAPAN-2 (purchased from Beina Chuanglian Biotechnology Co., Ltd., final sample concentration 0.006-100 nM), and human lung adenocarcinoma cell line HCC827 (purchased from Nanjing Kebai Biotechnology Co., Ltd., final sample concentration 0.012-50 nM). The results are shown in Figures 5-7. Antibody A-ADC had a significant killing effect on all tumor cells, while antibody A and iso-ADC had no significant killing effect.

[0417] 3.7 Detection of the bystander killing effect of antibody A-ADC on TROP2 and HER3 negative cells

[0418] To investigate the bystander effect of antibody A-ADC, BxPC-3 (TROP2, HER3 positive) single cell system, 293T-GFP (TROP2, HER3 negative) single cell system, and BxPC-3 and 293T-GFP (1:1) mixed cell system were treated with antibody A-ADC and iso-ADC, respectively. After 96 hours, cells in all sample wells were counted and analyzed by flow cytometry:

[0419] Single cell plating (mono-culture): The cell density of both cells was adjusted to 3×10 4 / mL, and 100 μL / well of each was plated in a 96-well plate, with 3×10 cells per well. 3 , cultured overnight in a 37°C, 5% CO2 incubator;

[0420] Co-culture: The two cell lines were mixed in culture medium (RPMI-1640 + 10% FBS), with the BxPC-3 cell density at 3.0 × 10 4 / mL, and the 293T-GFP cell density was 3.0×10 4 / mL; plate 96-well plates at 100 μL / well and culture overnight at 37°C in 5% CO2;

[0421] 100 μL of antibody A-ADC and iso-ADC at a working concentration of 10 nM was added to each well, and a non-drug control group (Negative Control) was set up at the same time. Each sample was repeated three times, and then cultured at 37 ° C, 5% CO2 for 96 hours. Subsequently, for the single-cell sample wells, 20 μL CCK-8 was added, and the OD value was detected by microplate reader at 450 / 650nm after being placed at 37 ° C for 3 hours. For the mixed cell sample wells, the cells were trypsinized, and after the cells were counted, the remaining cells were washed once with DPBS and then placed on the flow cytometer. The number of cells N was detected using a cell counter, and the flow cytometer detected the proportion of TROP2 and HER3 positive cells (V1L% parent population) in each concentration group in the mixed cell group, as well as the proportion of TROP2 and HER3 negative cells (V1R% parent population).

[0422] For single-cell systems:

[0423] For mixed cell systems:

[0424] The results are shown in Figure 8. In the single-cell system, 10 nM antibody A-ADC had an average inhibition rate of 65.31% against BxPC-3 cells, but had no significant killing effect on 293T-GFP cells. In the mixed system, 10 nM antibody A-ADC had an average inhibition rate of 43.86% against BxPC-3 cells and 78.8% against 293T-GFP cells. This indicates that compared to iso-ADC, antibody A-ADC has a significant bystander killing effect on TROP2- and HER3-negative cells.

[0425] 3.8 Evaluation of the anti-tumor effect of antibody A-ADC in a subcutaneous xenograft mouse model of human lung cancer CDX model HCC827

[0426] The animal model was established by subcutaneously injecting human HCC827 cells into BALB / c Nude mice (purchased from Weitonglihua (Beijing) Laboratory Animal Technology Co., Ltd.): 0.2 mL (10×10 6 HCC827 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 123 mm 3At 37 pm, animals were randomly divided into five groups based on body weight and tumor volume: vehicle control (PBS), iso-ADC group (10 mg / kg), low-dose Antibody A-ADC group (1 mg / kg), medium-dose Antibody A-ADC group (3 mg / kg), and high-dose Antibody A-ADC group (10 mg / kg). Each group consisted of six animals. Grouping was designated day 0, and the test substance was intraperitoneally injected on days 1, 5, 8, and 12, for a total of four doses. Tumor volume and mouse body weight were measured twice weekly during the experiment.

[0427] The results showed that during the trial, there was no significant difference in the body weight of the animals in the antibody A-ADC treatment group compared with the PBS group, and no obvious abnormalities were observed in the mice. As shown in Figure 9, 32 days after group administration, the average tumor volume of the vehicle control group was 1552 mm 3 The tumor volumes of the antibody A-ADC (1 mg / kg, 3 mg / kg and 10 mg / kg) treatment groups were 1073 mm 3 , 743mm 3 and 120mm 3 , TGI% (relative tumor inhibition rate, percentage value) were 33.4%, 56.5% and 100.2% respectively (p values ​​were 0.0103, 0.0001 and 0.0001 respectively).

[0428] As shown, in the CDX model of human lung cancer HCC827, Antibody A-ADC demonstrated significant tumor inhibition in a dose-dependent manner over a 1-10 mg / kg dose range over four administrations. Furthermore, body weight changes demonstrated that Antibody A-ADC was well tolerated at all doses.

[0429] 3.9 Evaluation of the anti-tumor effect of antibody A-ADC in the human gastric cancer CDX model NCI-N87 subcutaneous xenograft mouse model

[0430] 0.2 mL (10 × 10 6 NCI-N87 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated into the right back of each BALB / c nude mouse, and the average tumor volume reached approximately 169 mm 3 The animals were randomly divided into three groups according to body weight and tumor volume, namely, vehicle control group (PBS), antibody A-ADC low-dose group (3 mg / kg) and antibody A-ADC high-dose group (10 mg / kg), with 6 animals in each group. The drugs were administered once a week for a total of 2 weeks.

[0431] The results showed that the single-drug test groups of each test substance had no significant effect on the weight of the mice, and the mice showed no obvious abnormalities. As shown in Figure 10, 20 days after the start of administration, the average tumor volume of the tumor-bearing mice in the solvent control group reached 826 mm3 In the test antibody A-ADC (10 mg / kg, 3 mg / kg) treatment groups, the tumor volumes were 209 mm 3 and 591mm 3 (T / C%=27.22% and 72.64%; TGI%=94.07% and 35.65%; p<0.0001 and p=0.0807), among which the test antibody A-ADC (10 mg / kg) treatment group had a significant tumor inhibition effect compared with the control group.

[0432] 3.10 Evaluation of the anti-tumor efficacy and tolerability of Antibody A-ADC in the osimertinib-resistant human lung cancer PDX model LU-01-1377 subcutaneous xenograft NOD / SCID mouse model

[0433] Human tumor PDX models are initially created from surgically resected clinical specimens, which are designated as P0 upon implantation into experimental mice. P0 tumor tissue is then implanted into the next generation, designated P1. This process is repeated throughout the generations. FP3 tumors are revived from P2 generation tissue. The next generation from FP3 is designated FP4, and so on, through FP8 generation tissue, which will be used in the efficacy trials of the LU-01-1377 model.

[0434] 20~30mm 3 LU-01-1377 tumor tissue blocks were inoculated into the right back of each NOD / SCID mouse (purchased from Weitonglihua (Beijing) Laboratory Animal Technology Co., Ltd.) to allow tumor growth. In vivo efficacy experiments were conducted on the 33rd day after LU-01-1377 tumor block inoculation, and the average tumor volume reached 141 mm 3 Randomization and dosing began at 1:00 PM. The day of grouping was defined as Day 1, and dosing began immediately after grouping. The trial was divided into antibody A-ADC treatment groups (1 mg / kg, 3 mg / kg, and 10 mg / kg) and a vehicle PBS negative control group, with 6 animals in each group. After grouping, dosing was performed twice a week via tail vein injection. Efficacy and tolerability were evaluated based on relative tumor inhibition rate (TGI) and body weight change.

[0435] The results of weight changes showed that all animals in the control and test drug groups were alive on the 27th day after the first administration, with no significant weight loss. The anti-tumor efficacy results are shown in Figure 11. The average tumor volume of mice in the PBS control group on the 27th day after the first administration was 1244±216mm 3 The average tumor volume of the high-dose antibody A-ADC treatment group (10 mg / kg) was 0±0 mm on day 27 after the first administration. 3(10 mg / kg), the relative tumor inhibition rate TGI (%) was 112.78%, and there was a statistically significant difference in this treatment group compared with the PBS control group (p < 0.0001); the average tumor volume of the antibody A-ADC medium-dose treatment group (3 mg / kg) on ​​the 27th day after the first administration was 81 ± 43 mm 3 The relative tumor inhibition rate TGI (%) was 105.43%, which was statistically significant compared with the PBS control group (p < 0.0001). The average tumor volume of the low-dose A-ADC treatment group (1 mg / kg) was 1085 ± 171 mm on the 27th day after the first administration. 3 The relative tumor inhibition rate TGI (%) was 14.35%. Compared with the PBS control group, there was no statistically significant difference in this treatment group (p=0.7601).

[0436] 3.11 Evaluation of the anti-tumor effect of antibody A-ADC in a BALB / c nude female mouse model with subcutaneous transplantation of HCC827 cells

[0437] BALB / c nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously inoculated with 100 μL of 1×10 6 HCC827 cells were resuspended in a mixture of PBS and Matrigel (1:1) during inoculation. Tumor growth was observed regularly after inoculation. 3 The mice were randomly divided into groups according to tumor size and dosed at the same time, with the day of grouping defined as day 0. The trial was divided into an antibody A-ADC treatment group (11.5 mg / kg), a sacituzumab Govitecan positive control group (10 mg / kg and 20 mg / kg, purchased from Gilead Sciences, hereinafter referred to as SG), and a PBS blank control group. Each group consisted of 6 mice, and the drug was administered by tail vein injection. A total of four doses were administered once a week, and the experiment was terminated 49 days after the first dose. Efficacy was evaluated based on tumor growth inhibition rate, and safety was evaluated based on animal body weight changes and mortality.

[0438] At the end of the experiment, except for one mouse death in the SG 10 mg / kg group, no animal death was found in the other groups, and the weight of the mice did not decrease abnormally. The anti-tumor efficacy results are shown in Figure 12. The average tumor volume of the mice in the PBS group at the end of the experiment was 580 mm 3 The tumor volume of the antibody A-ADC treatment group (11.5 mg / kg) was 43 mm 3Compared with the PBS group, the TGI (%) was 92.6% (p < 0.001); the tumor volumes of the positive control SG 10 mg / kg group and 20 mg / kg group were 244 mm 3 and 121mm 3 , TGI (%) was 58.0% (p<0.05) and 79.2% (P<0.001) compared with the PBS group.

[0439] 3.12 Study on the Stability of Antibody A-ADC in Serum from Different Species

[0440] Antibody A-ADC was diluted with human, cynomolgus macaque, rat, and mouse serum to 10 μg / mL and 100 μg / mL, respectively. The aliquots were aliquoted into 100 μL tubes, with one aliquot prepared for each time point. The aliquots were incubated at 37°C for 0, 3, 7, 14, and 21 days, then removed and frozen. DXd levels in serum were determined using LC-MS / MS: DXd was extracted from the test samples by protein precipitation, and the processed samples were analyzed using liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0441] The results showed that by day 21, the release rates of DXd in human, cynomolgus macaque, rat, and mouse serum at 100 μg / mL of Antibody A-ADC were 0.637%, 0.698%, 0.355%, and 0.439%, respectively. The release rates of DXd in human, cynomolgus macaque, rat, and mouse serum at 10 μg / mL of Antibody A-ADC were 0.696%, 0.702%, 0.368%, and 0.492%, respectively. This demonstrates that Antibody A-ADC is stable in the serum of various species and exhibits a low shedding rate of the loaded toxin DXd.

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

Claims

1. A human epidermal growth factor receptor 3 (HER3) binding protein comprising at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:

1.

2. The HER3 binding protein of claim 1, wherein the CDR1, CDR2, and CDR3 are defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.

3. The HER3 binding protein of claim 1 or 2, wherein the immunoglobulin single variable domain is camelid, humanized, or chimeric.

4. The HER3 binding protein according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 8-10, SEQ ID NOs: 11-13, SEQ ID NOs: 14-16 and SEQ ID NOs: 17-19.

5. The HER3 binding protein according to any one of claims 1 to 4, wherein the at least one immunoglobulin single variable domain comprises one or more of the amino acid sequences shown in SEQ ID NOs: 1 to 7. The HER3 binding protein according to claim 5 , wherein the at least one immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-7.

7. The HER3 binding protein according to any one of claims 1 to 6, wherein the HER3 binding protein comprises one of the immunoglobulin single variable domains.

8. The HER3 binding protein of any one of claims 1-6, wherein the HER3 binding protein comprises 2, 3, 4, 5 or more of the immunoglobulin single variable domains, optionally, the 2, 3, 4, 5 or more of the immunoglobulin single variable domains have the same sequence or do not have the same sequence. 9 . The HER3-binding protein according to claim 1 , further comprising an immunoglobulin Fc region, preferably an Fc region of a human immunoglobulin, more preferably an Fc region of human IgG1, IgG2, IgG3 or IgG4. 10 . The HER3 binding protein according to claim 9 , wherein the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:

20. The HER3-binding protein according to claim 9 or 10, wherein the immunoglobulin Fc region is directly connected to the at least one immunoglobulin single variable domain or is indirectly connected to the at least one immunoglobulin single variable domain via a linker.

12. A multispecific antigen-binding protein comprising the HER3 binding protein or antigen-binding fragment thereof of claims 1-11 providing a first binding specificity.

13. The multispecific antigen-binding protein of claim 12, further comprising a second binding specificity provided by an antibody or antigen-binding fragment thereof that binds trophoblast cell surface antigen 2 (TROP2), and optionally further binding specificities.

14. A multispecific antigen-binding protein comprising an antibody or antigen-binding fragment thereof that binds to trophoblast cell surface antigen 2 (TROP2) and at least one immunoglobulin single variable domain that binds to human epidermal growth factor receptor 3 (HER3); Preferably, The antibody or antigen-binding fragment thereof that binds to TROP2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1, a VH CDR2, and a VH CDR3 selected from any one group of SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, and SEQ ID NOs: 31-33, The light chain variable region comprises a VL CDR1, a VL CDR2, and a VL CDR3 selected from any one group of SEQ ID NOs: 35-37, SEQ ID NOs: 38-40, SEQ ID NOs: 41-43, and SEQ ID NOs: 44-46; and / or Preferably, the HER3-binding immunoglobulin single variable domain comprises a VHH CDR1, a VHH CDR2, and a VHH CDR3 selected from any one group consisting of SEQ ID NOs: 8-10, SEQ ID NOs: 11-13, SEQ ID NOs: 14-16, and SEQ ID NOs: 17-19. 15 . The multispecific antigen-binding protein of claim 14 , wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises the amino acid sequence shown in SEQ ID NO:

21. 16 . The multispecific antigen-binding protein of claim 14 , wherein the light chain variable region of the antibody or antigen-binding fragment thereof that binds to TROP2 comprises the amino acid sequence shown in SEQ ID NO:

34.

17. The multispecific antigen-binding protein according to any one of claims 14 to 16, wherein the heavy chain of the TROP2-binding antibody or antigen-binding fragment thereof further comprises a human IgG1 constant region or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO:

47.

18. The multispecific antigen-binding protein according to any one of claims 14-17, wherein the light chain of the TROP2-binding antibody or antigen-binding fragment thereof further comprises a constant region of human Igκ or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO:

48. 19 . The multispecific antigen-binding protein of claim 14 , wherein the heavy chain of the TROP2-binding antibody comprises the amino acid sequence shown in SEQ ID NO:

49. 20 . The multispecific antigen-binding protein of any one of claims 14 to 19 , wherein the light chain of the TROP2-binding antibody comprises the amino acid sequence shown in SEQ ID NO:

50.

21. The multispecific antigen-binding protein of any one of claims 14-20, wherein the at least one immunoglobulin single variable domain that binds to HER3 comprises the amino acid sequence shown in one of SEQ ID NOs: 1-7.

22. The multispecific antigen-binding protein of claim 21, comprising one immunoglobulin single variable domain.

23. The multispecific antigen-binding protein according to any one of claims 14-22, wherein the at least one immunoglobulin single variable domain that binds HER3 is directly or indirectly linked via a linker to an antibody or antigen-binding fragment that binds TROP2.

24. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the C-terminus of the at least one immunoglobulin single variable domain that binds HER3 is connected to the N-terminus of the heavy chain of the antibody or antigen-binding fragment that binds TROP2.

25. The multispecific antigen-binding protein of any one of claims 14-23, wherein the N-terminus of the at least one immunoglobulin single variable domain that binds HER3 is linked to the C-terminus of the heavy chain of the antibody or antigen-binding fragment that binds TROP2.

26. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the C-terminus of the at least one immunoglobulin single variable domain that binds HER3 is connected to the N-terminus of the light chain of the antibody or antigen-binding fragment that binds TROP2.

27. The multispecific antigen-binding protein according to any one of claims 14-23, wherein the N-terminus of the at least one immunoglobulin single variable domain that binds HER3 is connected to the C-terminus of the light chain of the antibody or antigen-binding fragment that binds TROP2.

28. The multispecific antigen-binding protein of any one of claims 14-27, comprising a first polypeptide represented by SEQ ID NO: 51 and a second polypeptide represented by SEQ ID NO:

50.

29. A protein-drug conjugate having the structure of Formula I: P-(L1-sp1-L2-sp2-D)n (I), in, Protein P comprises the multispecific antigen-binding protein according to any one of claims 12 to 28, D is a biologically active substance, L1 is a linker for connecting to P, sp1 is a first spacer unit, L2 is a cleavable linker, sp2 is a second spacer unit and is connected to D, and n=1 to 20.

30. The protein-drug conjugate of claim 29, wherein L1 is selected from: in, Ar represents C 6-10 arylene, which is optionally substituted by halogen, C 1-6 Alkyl substituted; R1 is selected from hydrogen, halogen and C 1-6 Alkyl; Z is selected from a straight bond, C 2-6 Alkynylidene, C 2-6 Alkenylene, C 6-10 Arylene, 5-10 membered heteroarylene, amide, sulfonamide, imine and CF2.

31. The protein-drug conjugate according to claim 29 or 30, wherein the structure of sp1 is as shown in Formula II: in, a1=0 or 1, a2=an integer from 0 to 6, b1=0 or 1, b2=an integer from 0 to 16, b3=an integer from 0 to 16, c=an integer from 0 to 6, and at least one of b2 and b3 is 0.

32. The protein-drug conjugate of claim 31, wherein: (1) a1=0, a2=2, 3, 4, 5 or 6, b1=0, b2=0, b3=0, c=0; (2) a1=0, a2=0, b1=0, b2=0, b3=0, c=2, 3, 4, 5, or 6; (3) a1=1, a2=2, 3, 4, 5, or 6, b1=1, b2=2, 3, 4, 5, 6, 7, or 8, b3=0, c=0; (4) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; or (5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7 or 8, c = 2, 3, 4, 5 or 6.

33. The protein-drug conjugate according to any one of claims 29 to 32, wherein L2 is a dipeptide, tripeptide or tetrapeptide amino acid residue.

34. The protein-drug conjugate of claim 33, wherein L2 is a dipeptide amino acid residue selected from the group consisting of -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Val-Cit-, -Ala-Lys-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, -Gly-Gly-, -Ala-Ala-, -Gly-Val-, and -Gly-Glu-; the left side of the dipeptide amino acid residue is connected to sp1, and the right side is connected to sp2.

35. The protein-drug conjugate of claim 33, wherein L2 is a tripeptide amino acid residue selected from the group consisting of -Glu-Val-Ala-, -Glu-Val-Cit-, -αGlu-Val-Ala-, -αGlu-Val-Cit-, -Val-Lys-Gly, and -Val-Cit-Gly-; the left side of the tripeptide amino acid residue is connected to sp1, and the right side is connected to sp2.

36. The protein-drug conjugate of claim 33, wherein L2 is a tetrapeptide amino acid residue selected from the group consisting of -Gly-Gly-Phe-Gly- and -Gly-Phe-Gly-Gly-; the left side of the tetrapeptide amino acid residue is connected to sp1, and the right side is connected to sp2.

37. The protein-drug conjugate of any one of claims 29-36, wherein sp2 is absent, or sp2 is selected from: R2 is independently selected from hydrogen, C 1-6 Alkyl, hydroxy, amino, halogen, nitro, cyano, d is an integer from 1 to 20, e is an integer from 1 to 20; R3 and R4 are each independently selected from hydrogen and C 1-6 alkyl; The alkyl group may be optionally substituted with hydroxy, amino, halogen, nitro and cyano.

38. The protein-drug conjugate according to any one of claims 29 to 37, wherein the -L1-sp1-L2-sp2- is selected from the following structures: k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

39. The protein-drug conjugate of any one of claims 29-38, wherein D is selected from the group consisting of a cytotoxin, a protein kinase inhibitor, an immunostimulant, a glucocorticoid, an oligonucleotide, a radioisotope, a polypeptide, and any combination thereof.

40. The protein-drug conjugate of claim 39, wherein D is a cytotoxin selected from the group consisting of DNA alkylating agents, DNA destructors, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, ribosome inhibitors, and any combination thereof.

41. The protein-drug conjugate of claim 40, wherein D is selected from the group consisting of: auristatin derivatives, maitansine derivatives, eribulin derivatives, tubulysin derivatives, pyrrolobenzodiazepine (PDB) derivatives, duocarmycin derivatives, calicheamicin derivatives, PNU-159682 and its derivatives, camptothecin derivatives, amatoxin derivatives, and any combination thereof.

42. The protein-drug conjugate of claim 40, wherein D has a structure represented by Formula III: in, X is selected from CH2, NH, O, S or SO2; Y does not exist, or Y has The structure shown; wherein W1 and W3 are each independently selected from O, S and NH, and W2 is selected from CH and N, R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano and nitro; or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl or a carbonyl group; or, R a is connected to the N atom of the amide portion to form a 3-6 membered heterocycloalkyl group and R b is hydrogen; Ring A is selected from the group consisting of a 5-10 membered cycloalkylene, a 5-10 membered heterocycloalkylene, a 6-10 membered arylene, and a 5-10 membered heteroarylene; The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano, carbonyl and nitro; d and e are each independently selected from integers from 0 to 5.

43. The protein-drug conjugate of claim 42, wherein D has a structure represented by formula III-a: Where W1 is O or NH; R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxy, amino, cyano and nitro; or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl or a carbonyl group; or, R a is connected to the N atom of the amide portion to form a 3-6 membered heterocycloalkyl group and R b is hydrogen; The alkyl, alkoxy, cycloalkyl and heterocycloalkyl groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxy, amino, cyano and nitro; d is an integer from 0 to 5.

44. The protein-drug conjugate according to claim 43, wherein W1 is O.

45. The protein-drug conjugate of claim 43 or 44, wherein d is 0, 1 or 2.

46. ​​The protein-drug conjugate according to any one of claims 43-45, wherein R a Selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1- 6 alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, hydroxyl and amino, R b For hydrogen.

47. The protein-drug conjugate according to any one of claims 43-45, wherein R a and R b Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group or a 3-6 membered heterocycloalkyl group.

48. The protein-drug conjugate of claim 43, wherein D is selected from the group consisting of:

49. The protein-drug conjugate of claim 42, wherein D has the structure shown in Formula III-b: Where W3 is O or NH, W2 is CH and N, Ring A is selected from the group consisting of a 5-10 membered cycloalkylene, a 5-10 membered heterocycloalkylene, a 6-10 membered arylene, and a 5-10 membered heteroarylene; The cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are each independently optionally further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl and nitro; e is an integer from 0 to 5.

50. The protein-drug conjugate according to claim 49, wherein W3 is O.

51. The protein-drug conjugate according to claim 49 or 50, wherein W2 is CH.

52. The protein-drug conjugate according to any one of claims 49 to 51, wherein e is 0, 1, or 2.

53. The protein-drug conjugate according to any one of claims 49 to 52, wherein Ring A is a 5- to 10-membered cycloalkylene group.

54. The protein-drug conjugate of claim 49, wherein D is selected from the group consisting of:

55. The protein-drug conjugate of claim 41, wherein D is a camptothecin derivative; preferably selected from the following structures:

56. The protein-drug conjugate of claim 29, wherein the -L1-sp1-L2-sp2-D is selected from the following structures: k is an integer from 1 to 20.

57. The protein-drug conjugate of any one of claims 29-56, wherein L1 is linked to the multispecific antigen-binding protein P via a sulfhydryl group, wherein the sulfhydryl group is obtained by reducing the disulfide bonds between heavy chains and / or the disulfide bonds between heavy chains and light chains. The protein-drug conjugate according to any one of claims 29 to 56 , wherein L1 is linked to the multispecific antigen-binding protein P via an oligosaccharide.

59. The protein-drug conjugate of claim 58, wherein the oligosaccharide has a structure as shown in Formula Va or Formula Vb: in, P* binds to HER3 and TROP2, comprising the antigen binding protein of any one of claims 12-28, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20; Gal* is a modified galactose selected from the following structures: The oligosaccharide is linked to P* via a core GlcNAc. The protein-drug conjugate of claim 59 , wherein the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

61. The protein-drug conjugate of any one of claims 58 to 60, wherein the oligosaccharide is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to Asn297 (numbering according to the EU index of Kabat) of the Fc fragment.

62. The protein-drug conjugate according to any one of claims 29 to 61, wherein the protein-drug conjugate has a structure shown in Formula VI: in, P* binds to HER3 and TROP2, for example, comprising the antigen binding protein of any one of claims 12-28, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20; Gal* is a modified galactose selected from the following structures: The oligosaccharide is linked to P* via a core GlcNAc; LP is selected from the following structures: (a) and / or (b) and / or (c) and / or as well as (d) and / or k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Among them, the core GlcNAc is directly connected to P*.

63. The protein-drug conjugate according to any one of claims 59 to 62, wherein the antigen binding protein comprises a first polypeptide represented by SEQ ID NO: 51 and a second polypeptide represented by SEQ ID NO:

50.

64. The protein-drug conjugate of claim 62 or 63, wherein the oligosaccharide is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to Asn297 (numbering according to the EU index of Kabat) of the Fc fragment.

65. A protein derivative having the structure shown in Formula VII: in, P* binds to HER3 and TROP2, comprising the antigen binding protein of any one of claims 12-28, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and j is 1 to 20; Gal** is a modified galactose selected from the following structures: The oligosaccharide is linked to P* via a core GlcNAc.

66. The protein derivative according to claim 65, wherein the core GlcNAc is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to Asn297 (numbering according to the EU index of Kabat) of the Fc fragment.

67. An isolated polynucleotide encoding the HER3 binding protein or antigen-binding fragment thereof of any one of claims 1-11 or the multispecific antigen-binding protein of any one of claims 12-28.

68. A vector comprising the polynucleotide of claim 67, preferably said vector is an expression vector.

69. A host cell comprising the polynucleotide of claim 67 or the vector of claim 68, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (for example, the host cell is a CHO cell, such as a CHO-K1 cell or an expiCHO cell, or the host cell is a 293 cell, such as a HEK293 cell) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

70. A method for preparing a HER3 binding protein or an antigen binding fragment thereof or a multispecific antigen binding protein comprising a HER3 binding protein or an antigen binding fragment thereof, the method comprising culturing a host cell comprising a nucleic acid encoding the HER3 binding protein or an antigen binding fragment thereof of any one of claims 1 to 11 or the multispecific antigen binding protein of any one of claims 12 to 28 under conditions suitable for expressing the protein, optionally further comprising recovering the binding protein or the multispecific antigen binding protein or its antigen binding fragment or fusion protein from the host cell.

71. Use of the HER3 binding protein of any one of claims 1-11, the multispecific antigen binding protein of any one of claims 12-28, or the protein-drug conjugate of any one of claims 29-64 in the preparation of a medicament for treating and / or preventing tumors.

72. The use according to claim 71, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

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