Composition containing pd-l1 binding molecule and bispecific antibody-drug conjugate and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

Compositions comprising PD-L1 binding molecules and bispecific antibody-drug conjugates and uses thereof Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a composition comprising a PD-L1 binding molecule and a bispecific antibody-drug conjugate. Background Art

[0002] Antibody drugs are widely used in tumor treatment. Because antibodies can selectively bind to antigens or antigen-positive cells, they exhibit low off-target toxicity. However, antibody drugs alone are usually not enough to effectively fight tumors. Antibody-drug conjugates (ADCs) combine the advantages of antibodies and cytotoxins, enhancing the effectiveness and selectivity of tumor cell killing. Since the approval of the first ADC drug, Mylotarg, in 2000, 15 ADC drugs have been approved for marketing, and more ADC drugs targeting different targets and using different conjugation technologies are in the clinical research stage.

[0003] Bringing clinically effective ADCs to market requires the development of pharmaceutical formulations suitable for storage, transportation, and administration. However, proteins are susceptible to chemical and physical degradation, and small molecule conjugation introduces additional complexity and instability to antibodies. Marketed ADCs are generally administered at concentrations below 20 mg / mL, in stark contrast to the concentrations above 50 mg / mL used in traditional antibody therapies. This lower concentration reduces the risk of aggregation, particularly for drugs carrying hydrophobic payloads. Existing ADCs are typically administered via intravenous infusion. The low ADC concentration in the infusion bag increases the risk of drug loss due to adsorption to the plastic. Dilution of the drug also reduces stabilizer levels, potentially leading to aggregation and particle formation. Furthermore, intravenous infusion typically requires a prolonged administration period. Liquid formulations are generally less expensive to produce and more convenient to use than lyophilized formulations. However, due to product complexity and stability concerns, they can often only be formulated and used at lower concentrations, requiring lyophilized powders for long-term storage. Currently approved ADCs are stored and shipped in lyophilized form and reconstituted prior to use.

[0004] Members of the HER receptor tyrosine kinase family are important mediators of cell growth, differentiation, and survival. This receptor family includes four unique members, including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3), and HER4 (ErbB4 or tyro2). Members of this receptor family have been implicated in various types of human malignancies. Prior art has developed bispecific antibody-drug conjugates directed against HER2.

[0005] Programmed death-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The original members of this family, CD28 and ICOS, were discovered through their ability to enhance T cell proliferation after the addition of monoclonal antibodies. Two cell surface glycoprotein ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation and cytokine secretion after binding to PD-1. PD-L1 (B7-H1) and PD-L2 (B7-DC) are both B7 homologs that can bind to PD-1 but not to other CD28 family members. PD-L1 expression on the cell surface has also been shown to be upregulated by IFN-γ stimulation.

[0006] PD-L1 expression has been found in several murine and human cancers, including lung, ovarian, colon, melanoma, and various myelomas. Existing results indicate that high expression of PD-L1 by tumor cells plays a key role in tumor immune evasion by increasing T cell apoptosis. Researchers have found that the P815 tumor cell line transfected with the PD-L1 gene resists lysis by specific CTLs in vitro and exhibits enhanced tumorigenicity and invasiveness when inoculated into mice. These biological properties can be reversed by blocking PD-L1. In mice with PD-1 gene knockout, which blocks the PD-L1 / PD-1 pathway, inoculation with tumor cells prevents tumor formation. It has also been suggested that PD-L1 may be involved in intestinal mucosal inflammation, and that inhibition of PD-L1 prevents the atrophic disease associated with colitis. Patent application WO2017020801 discloses a class of single-domain antibodies targeting PD-L1 and their anti-tumor uses.

[0007] Given the structure and instability of ADC drugs developed in the prior art, there is a need to develop technologies to improve the stability of ADC drugs, and / or to obtain formulations containing ADC drugs suitable for long-term stable storage, such as high-concentration injections. Summary of the Invention

[0008] The inventors unexpectedly discovered that single-domain antibody molecules, such as anti-PD-L1 single-domain antibodies, can improve the stability of ADC drugs. For example, after combining single-domain antibody molecules (such as anti-PD-L1 single-domain antibodies) with ADC drugs, the stability of ADC drugs is significantly improved, and they can be formulated into high-concentration injections and stored stably for a long time. High-concentration injections can be used for subcutaneous injection, which greatly facilitates the clinical use of patients and also creates convenience for the combination of ADC drugs and immunotherapy.

[0009] In a first aspect, the present invention relates to the use of antigen (such as PD-L1) binding molecules in improving the stability of protein-drug conjugate formulations.

[0010] In a second aspect, the present invention also relates to a composition comprising an antigen (e.g., PD-L1) binding molecule and a protein-drug conjugate. The composition may be in the form of a solid preparation, such as a lyophilized preparation, or a liquid preparation, such as an injection, and may further comprise suitable pharmaceutical excipients. The composition is suitable for clinical use, such as subcutaneous injection.

[0011] In a third aspect, the present invention also relates to a method for treating advanced or metastatic solid malignant tumors, comprising administering a drug combination of an antigen (such as PD-L1) binding molecule and a protein-drug conjugate to a patient in need thereof.

[0012] 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

[0013] The accompanying drawings are briefly described as follows:

[0014] Figure 1A, B, and C show the insoluble particulate matter of JSKN003 alone and its mixture with KN035.

[0015] Figure 2 shows the aggregate content of JSKN003 alone and its mixture with KN035.

[0016] Figure 3 shows the changes in the drug concentration of JSKN003 in the sample serum after intravenous injection or subcutaneous injection; ◆ represents intravenous injection and ▲ represents subcutaneous injection.

[0017] Figure 4 shows the MS spectrum of LP1.

[0018] FIG5 shows the reaction formula for preparing antibody A-(N3)4 and the structure of UDP-GalNAz.

[0019] FIG6 shows the mass spectrometry data of antibody A-(N3)4.

[0020] FIG7 shows the reaction formula for preparing JSKN003.

[0021] FIG8 shows the mass spectrometry data of JSKN003.

[0022] Figure 9 shows the insoluble particles of JSKN003 alone and mixed with KN035 after 4 weeks.

[0023] Figures 10A-10C show the results of the SEC-HPLC test of the relative binding activity of the aggregate (A), nrCE-SDS fragment (B) and antibody portion (C) in the high temperature stability experiment in the first round of formulation screening.

[0024] Figures 11A-11F show the test results of insoluble particles (A), SEC-HPLC aggregates (B, only FS2-1SEC-HPLC test results were obtained at T0, and this data was used as a reference), nrCE-SDS fragments (C), CEX-HPLC main peaks (D) and relative binding activity of the antibody portion (E) in the second round of formulation screening, as well as the viscosity (F) test results in the second round of formulation screening.

[0025] Figures 12A-12E show the test results of insoluble particles (A), SEC-HPLC aggregates (B, no SEC-HPLC data was obtained at T0, and the test results of the sample before liquid exchange were used as a reference), nrCE-SDS fragments (C), CEX-HPLC main peak (D) and relative binding activity of the antibody portion (E) in the third round of prescription screening.

[0026] Figure 13 shows the main peak detection results of insoluble particles, SEC-HPLC and CEX-HPLC in the excipient content confirmation experiment.

[0027] Figures 14A-14D show the results of long-term stability experiments of the mixed formulations, including: SEC-HPLC (A), CEX-HPLC (B), nrCE-SDS (C) and relative binding activity (D).

[0028] Figures 15A-15D show the results of accelerated stability experiments of mixed formulations, including: SEC-HPLC (A), CEX-HPLC (B), nrCE-SDS (C) and relative binding activity (D).

[0029] Figure 16A, B, C, D, E and F show the insoluble particulate content of DS8201 alone and its mixed formulation with KN035.

[0030] Figures 17A and 17B show the aggregate content of the DS8201 mixed formulation by SEC-HPLC.

[0031] Figure 18A, B and C show the insoluble particulate content of RC48 alone and its mixed formulation with KN035.

[0032] FIG19 shows the aggregate content of the RC48 mixed formulation by SEC-HPLC. DETAILED DESCRIPTION

[0033] Definition of terms

[0034] 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.

[0035] 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.

[0036] "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)).

[0037] In the present invention, the regions of CDR defined by Kabat, AbM or IMGT schemes are as follows:

[0038] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0039] 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.

[0040] 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.

[0041] In the present 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 one embodiment of the present application, the light chain of the antibody that can specifically bind to the first antigen or antigenic epitope and the light chain of the antibody that can specifically bind to the second antigen or antigenic epitope in the bispecific antibody have the same sequence. In one embodiment of the present application, the heavy chain of the antibody that can specifically bind to the first antigen or antigenic epitope and the heavy chain of the antibody that can specifically bind to the second antigen or antigenic epitope in the bispecific antibody have different sequences.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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. Immunoglobulin variable domains confer specificity to antibodies for antigens by having antigen-binding sites.

[0047] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. One example of an immunoglobulin single variable domain within the meaning of this application is a "domain antibody," such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae, as defined below.

[0048] "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). A VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. VHHs are, for example, derived from camelids, such as alpacas, or are humanized or sequence-optimized forms thereof (e.g., affinity-matured forms to increase binding affinity). In some embodiments, a VHH is a monovalent, monospecific polypeptide molecule consisting of or essentially consisting of a single heavy chain variable region (e.g., the heavy chain variable region of a heavy chain antibody).

[0049] In the context of this application, the terms "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0050] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), 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)).

[0051] 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.

[0052] 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).

[0053] 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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (SwissProt P04626). In this application, the HER2 may also be referred to as rbB-2, NEU, HER-2, or CD340. The HER2 may include any variant, isoform, and species homolog of HER2 that is naturally expressed by cells, including tumor cells, or expressed by cells transfected with a HER2 gene or cDNA.

[0059] 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.

[0060] 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.

[0061] The term "cycloalkylene" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon radical derived from a parent alkane by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms.

[0062] 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.

[0063] The term "heterocycloalkylene" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon radical 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.

[0064] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0072] As used herein, the term "and / or" refers to any one of the alternatives or two or more of the alternatives.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] "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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] As used herein, "treat," ...

[0084] Detailed Description of the Invention

[0085] Improving the stability of protein-drug conjugate formulations

[0086] In a first aspect, the present application provides the use of antigen-binding molecules to improve the stability of protein-drug conjugate formulations. The stability includes but is not limited to conformational stability, colloidal stability, high temperature stability, freeze-thaw stability, shaking stability, light stability, and long-term storage stability.

[0087] In some embodiments, the preparation is a solid preparation, preferably a lyophilized preparation.

[0088] In some embodiments, the preparation is a liquid preparation, preferably a solution, more preferably an injection, such as a subcutaneous injection or an intravenous injection.

[0089] In some embodiments, the antigen binding molecules are capable of reducing the amount of insoluble particulates produced in the formulation.

[0090] In some embodiments, the antigen binding molecules are capable of reducing the amount of aggregates formed in a formulation.

[0091] In some embodiments, the antigen binding molecule is a programmed death-ligand 1 (PD-L1) binding molecule.

[0092] In some embodiments, the PD-L1 binding molecules of the present invention are derived from WO2017020801, which is incorporated herein in its entirety.

[0093] In some embodiments, the PD-L1 binding molecule of the present invention comprises the CDR1, CDR2 and CDR3 of the VHH disclosed in WO2017020801, or comprises the VHH disclosed therein, or comprises the heavy chain antibody disclosed therein, or consists thereof.

[0094] In some embodiments, the PD-L1 binding molecule comprises or consists of an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 of the VHH set forth in SEQ ID NO: 1. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

[0095] In some embodiments, the CDR1, CDR2, and CDR3 in the VHH set forth in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 5-7 (Kabat), SEQ ID NOs: 8-10 (AbM), SEQ ID NOs: 11-13 (Chothia), and SEQ ID NOs: 14-16 (IMGT).

[0096] In some embodiments, the immunoglobulin single variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. Preferably, the amino acid sequence of the immunoglobulin single variable domain is as shown in SEQ ID NO: 1.

[0097] In some embodiments, the PD-L1 binding molecule is a PD-L1 heavy chain antibody, which comprises VHH and an antibody constant region or a portion thereof, such as an Fc region, for example, a heavy chain antibody comprising VHH-constant region or VHH-CH1-Fc or VHH-Fc.

[0098] Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region, such as the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or variants thereof.

[0099] In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0100] In some embodiments, the VHH can be located at the N-terminus of the immunoglobulin Fc region, or at the C-terminus of the immunoglobulin Fc region; the two are directly connected or connected through a peptide linker.

[0101] In some embodiments, the aforementioned peptide chain is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(GGGS) n 、(GGSG)n 、(GGGGS) n and (GAP) n , wherein n is an integer from 1 to 10.

[0102] In some embodiments, the PD-L1 binding molecule comprises the amino acid sequence shown in SEQ ID NO: 4; preferably, the amino acid sequence of the PD-L1 binding molecule is shown in SEQ ID NO: 4.

[0103] In one embodiment, the PD-L1 heavy chain antibody according to the present invention can dimerize with another polypeptide chain comprising an Fc region (e.g., the same or different heavy chain antibody) through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising the PD-L1 heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a PD-L1 heavy chain antibody formed by pairing two identical PD-L1 heavy chain antibody chains.

[0104] In some embodiments, the protein-drug conjugate has the structure of Formula I: P-(LD) n (I),

[0105] Wherein, P is a protein, L is a linking unit, D is a substance with biological activity; and n is an integer from 1 to 20.

[0106] In some embodiments, the protein P has desired physiological properties, and preferably, the protein P can prevent and / or treat a disease or condition. Specifically, the protein P can be an antibody, a fusion protein, a cytokine, a polypeptide, and the like.

[0107] In some embodiments, the protein P is an antibody; the antibody targets one or more of the following antigens: 4-1BB, 4-1BBL, A33, adenosine A2a receptor, Akt, ALK, androgen receptor, Ang-1, Ang-2, Annexin A3, Aurora A, Aurora B, B7-H3, B7-H4, Bcl-2, Bcr-Abl, BRAF, BTK, BTLA, BTN2A1, CA-125, CAIX, CCR4, CD105 / endoglin, CD109, CD123, CD155, CD16, CD160, CD19, CD20, CD200, CD200R, CD22, CD24 , CD25, CD27, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD40L, CD47, CD48, CD52, CD70, CD79b, CD80, CD86, CD96, CDK4, CDK6, CDK9, CEA, CEACAM1, ChK1, ChK2, c-KIT, c-Met, C OX2, CSF-1R, CSF2, CTLA-4, CXCR2, CXCR4, DDR2, DLL3, DLL4, DNAM-1, DR5, EGFR, EpCAM, EPHA3, EphB4, ERK1, ERK2 / p38MAPK, FAK, FAP, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, Flt -3, Gal-9, GITR, GITRL, Glypican-3, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HER2, HER3, HER4 / ERBB4, HGF, HHLA2, HIF-1α, HSP27, HSP90, HVEM, ICOS, ICOS Ligand, IDO, IGF1R, IL-13, IL-6, JAK1, JAK2, JAK3, KRAS, LAG-3, LIGHT, MDM2, MEK1, MEK2, MMP-1, MMP-10, MMP-11, MMP-13, MMP-2, MMP-7, MMP-9, mTOR, Mucin1. Myc, NF-κB, NKG2A, NRAS, NTRK1, NTRK2, NTRK3, OX40, OX40L, p53, PAF, PARP1, PARP2, PD1, PDGFR-α, PDGFR-β, PD-L1, PD-L2, PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ, PIM1 , PIM3, PSMA, PTEN, RAF-1, RANKL, RET, S100A4, SIRPα, SLAMF7, SMO, Src, STAT3, STEAP-1, SYK, TDO, TGFβ, Tie-2, TIGIT, TIM-3, TLR8, TMIGD2, TNF-α, Toll-like receptor 3. TRAIL, TRAILR1, TROP-2, VEGF, VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3 and VISTA.

[0108] In some embodiments, the antibody can target HER2; preferably, the antibody can bind to different epitopes of human HER2, such as the extracellular domain II of human HER2, and / or the extracellular domain IV of human HER2;

[0109] In some embodiments, the antibody is a bispecific antibody targeting different epitopes of HER2. In some embodiments, the antibody can specifically bind to extracellular domain II and extracellular domain IV of human HER2.

[0110] In some embodiments, the bispecific antibody is derived from WO2016110267, which is incorporated herein in its entirety.

[0111] In some embodiments, the bispecific antibody comprises the bispecific structure shown in WO2016110267.

[0112] In some embodiments, the bispecific antibody comprises 1-6 CDRs of the first heavy chain, 1-6 CDRs of the second heavy chain, and 1-6 CDRs of the common light chain of the bispecific antibody described in WO2016110267.

[0113] In some embodiments, the bispecific antibody comprises the heavy chain variable region and / or light chain variable region of the first heavy chain, the heavy chain variable region and / or light chain variable region of the second heavy chain, and the heavy chain variable region and / or light chain variable region of the common light chain of the bispecific antibody described in WO2016110267.

[0114] In some embodiments, the bispecific antibody comprises the first heavy chain, the second heavy chain, and the common light chain of the bispecific antibody described in WO2016110267.

[0115] In some embodiments, the structure of the antibody is like a full-length antibody, but the two heavy chains are different, i.e., it has a first heavy chain, a second heavy chain and two common light chains. In some embodiments, the first heavy chain comprises a first heavy chain variable region, the second heavy chain comprises a second heavy chain variable region, and / or the common light chain comprises a common light chain variable region.

[0116] In some embodiments, the antibody comprises a first heavy chain, a second heavy chain, and a common light chain, wherein the first heavy chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 17, the second heavy chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 18, and the common light chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 19. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

[0117] In some embodiments, the CDR1, CDR2, and CDR3 in the heavy chain variable region of SEQ ID NO: 17 are selected from any one of the following groups: SEQ ID NOs: 23-25 ​​(Kabat), SEQ ID NOs: 26-28 (AbM), SEQ ID NOs: 29-31 (Chothia), and SEQ ID NOs: 32-34 (IMGT).

[0118] In some embodiments, the CDR1, CDR2, and CDR3 in the heavy chain variable region of SEQ ID NO: 18 are selected from any one of the following groups: SEQ ID NOs: 35-37 (Kabat), SEQ ID NOs: 38-40 (AbM), SEQ ID NOs: 41-43 (Chothia), and SEQ ID NOs: 44-46 (IMGT).

[0119] In some embodiments, the CDR1, CDR2, and CDR3 in the light chain variable region set forth in SEQ ID NO: 19 are selected from any one of the following groups: SEQ ID NOs: 47-49 (Kabat), SEQ ID NOs: 50-52 (AbM), SEQ ID NOs: 53-55 (Chothia), and SEQ ID NOs: 56-58 (IMGT).

[0120] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, or consists of the amino acid sequence.

[0121] In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, or consists of the amino acid sequence.

[0122] In some embodiments, the common light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, or consists of the amino acid sequence.

[0123] In some embodiments, the antibody comprises a first heavy chain, a second heavy chain, and two common light chains, wherein the first heavy chain comprises a first heavy chain variable region, the second heavy chain comprises a second heavy chain variable region, and the common light chains each comprise a light chain variable region, wherein

[0124] The first heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0125] The first heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18; and

[0126] The light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0127] In some embodiments, the amino acid sequence of the first heavy chain of the antibody comprises, or consists of, the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0128] In some embodiments, the amino acid sequence of the second heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consists of the amino acid sequence.

[0129] In some embodiments, the amino acid sequence of the common light chain of the antibodies comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consists of the amino acid sequence.

[0130] In some embodiments, the antibody comprises a first heavy chain, a second heavy chain, and two common light chains, wherein

[0131] The first heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 20;

[0132] The first heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; and

[0133] The light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 22.

[0134] In some embodiments, the linker L in Formula I has a structure shown in Formula II: -L1-sp1-L2-sp2- (II)

[0135] Among them, L1 is a linker for connecting to P, sp1 is the first spacer unit, L2 is a cleavable linker, and sp2 is the second spacer unit and is connected to D.

[0136] In some embodiments, the linker L in Formula I has a structure shown in Formula II: -L1-sp1-L2-sp2- (II)

[0137] Wherein, L1 is a linker for connecting to P, sp1 is the first spacer unit, L2 does not exist, and sp2 is the second spacer unit and is connected to D.

[0138] In some embodiments, in Formula II, L1 is selected from:

[0139] (The side connected to the protein is marked as P, and the side connected to the first spacer unit is marked as sp1),

[0140] Among them, 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.

[0141] In some embodiments, L1 is

[0142] In some embodiments, the structure of the first spacer unit sp1 in Formula II is:

[0143] (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.

[0144] In some embodiments, in the structure of sp1, a1=1; in other embodiments, a1=0.

[0145] In some embodiments, a2=0, 1, 2, 3, 4, 5, or 6.

[0146] In some embodiments, b1=0 or 1.

[0147] In some embodiments, b2=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0148] In some embodiments, b3=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0149] In some embodiments, c=0, 1, 2, 3, 4, 5, or 6.

[0150] 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.

[0151] In some specific embodiments, the structure of sp1 is selected from the following group:

[0152] (1) a1=0, a2=2, 3, 4, 5 or 6, b1=0, b2=0, b3=0, c=0;

[0153] (2) a1=0, a2=0, b1=0, b2=0, b3=0, c=2, 3, 4, 5, or 6;

[0154] (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;

[0155] (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

[0156] (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;

[0157] In some embodiments, the cleavable linker L2 in Formula II is a dipeptide, tripeptide, or tetrapeptide residue.

[0158] 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-.

[0159] 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.

[0160] 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.

[0161] In some embodiments, sp2 in Formula II is absent, or sp2 is selected from:

[0162] (The side connected to L2 is marked as L2, and the side connected to the biologically active substance D is marked as D), wherein,

[0163] 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;

[0164] The alkyl group may be optionally substituted with hydroxy, amino, halogen, nitro and cyano.

[0165] In some embodiments, sp2 is

[0166] In some embodiments, sp2 is

[0167] 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.

[0168] In some embodiments, the linking unit L can be selected from the following structures:

[0169] k and k' are each independently an integer of 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (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).

[0170] In some embodiments, k=2, 4, or 8. In some embodiments, k'=1, 2, 3, 4, 5, or 6.

[0171] In some embodiments, the linking unit L can be selected from the following structures:

[0172] k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (the side connected to protein P is marked as P, and the side connected to biologically active substance D is marked as D). In some embodiments, k=2, 4 or 8.

[0173] The biologically active substance D in formula I can be selected from cytotoxins, protein kinase inhibitors, immune agonists, glucocorticoids, oligonucleotides, radioisotopes, polypeptides and any combination thereof.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] In some embodiments, D has the structure shown in Formula III:

[0178] wherein X is selected from CH2, NH, O, S or SO2;

[0179] Y does not exist, or Y has The structure shown;

[0180] wherein W1 and W3 are each independently selected from O, S and NH, and W2 is selected from C, CH and N,

[0181] 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;

[0182] 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;

[0183] 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;

[0184] d and e are each independently selected from integers from 0 to 5.

[0185] In some embodiments, D has the structure shown in Formula III-a:

[0186] Where W1 is O or NH;

[0187] R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 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;

[0188] 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;

[0189] d is an integer from 0 to 5.

[0190] In some embodiments, W1 is O.

[0191] In some embodiments, d is 0, 1, or 2.

[0192] In some embodiments, wherein R a Selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 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.

[0193] In some embodiments, wherein R a and R bTogether with the carbon atoms to which it is attached, it forms 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, hydroxyl, amino, cyano and nitro. In some embodiments, D can be selected from the following groups:

[0194] In other embodiments, D has the structure shown in Formula III-b:

[0195] Where W3 is O or NH, W2 is C, CH or N,

[0196] 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;

[0197] 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;

[0198] e is an integer from 0 to 5.

[0199] In some embodiments, W3 is O; in other embodiments, W2 is CH.

[0200] In some embodiments, e is 0, 1, or 2.

[0201] 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.

[0202] In some embodiments, D can be selected from the group consisting of:

[0203] In some embodiments, D, as a camptothecin derivative, can also be selected from the following structures:

[0204] (i.e. Exatecan), (i.e. SN-38).

[0205] In some embodiments, D may be an auristatin derivative, preferably selected from the following structures:

[0206] In some embodiments, D may be a maytansine derivative, preferably selected from the following structures:

[0207] In some embodiments, D is eribulin and its derivatives, preferably selected from the following structures:

[0208] In some embodiments, in Formula I, the linker L is connected to the antibody via a thiol group; preferably, the thiol group is derived from the 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.

[0209] In other embodiments, the linker unit L is linked to the antibody via an oligosaccharide; preferably, the oligosaccharide is derived from the natural sugar chain of the antibody.

[0210] In some embodiments, the oligosaccharide is derived from an N-glycan chain of an antibody.

[0211] In some embodiments, the oligosaccharide consists of 2 to 15 monosaccharides; preferably, the oligosaccharide consists of 2 to 10 monosaccharides.

[0212] In some embodiments, the oligosaccharide has the structure shown in Formula Va or Formula Vb:

[0213] Wherein, P* is an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 20;

[0214] Gal* is a modified galactose selected from the following structures:

[0215] The oligosaccharide is linked to the antibody via a core GlcNAc.

[0216] In some embodiments, the modified galactose is linked to the GlcNAc via a β-1,4-glycosidic bond.

[0217] In some embodiments, the oligosaccharide is linked to the Fc fragment of the antibody; 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.

[0218] In some specific embodiments, the protein-drug conjugate described herein has a structure shown in Formula VI:

[0219] wherein P* is an antibody (the antibody may be as defined above), GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 10;

[0220] Gal* is a modified galactose selected from the following structures: LP is as defined above for LD; the core GlcNAc is directly linked to the antibody.

[0221] In some embodiments, LP is selected from one of the following structures (a) to (g):

[0222] (a) and / or

[0223] (b) and / or

[0224] (c) and / or

[0225] (d) and / or

[0226] (e) and / or

[0227] (f) and / or as well as

[0228] (g) and / or k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0229] In some embodiments, m is 1 to 4; in some embodiments, the DAR value of the conjugate as a whole is 1 to 8.

[0230] In some embodiments, the antibody comprises a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 20, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 21, and a common light chain having an amino acid sequence as shown in SEQ ID NO: 22.

[0231] Composition

[0232] In a second aspect, the present application also provides a composition comprising an antigen binding molecule and a protein-drug conjugate.

[0233] In some embodiments, the composition is a solid preparation, preferably a lyophilized preparation.

[0234] In some embodiments, the composition is a liquid preparation, preferably a solution, more preferably an injection, such as a subcutaneous injection or an intravenous injection.

[0235] The definitions of the antigen-binding molecules and protein-drug conjugates in the composition can refer to the technical features described in the first aspect. In addition, the following further definitions can be made:

[0236] In some embodiments, the antigen binding molecule comprises a VHH that binds to PD-L1, and the VHH that binds to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0237] In some embodiments, the PD-L1 binding molecule is a heavy chain antibody comprising an immunoglobulin Fc region.

[0238] In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or variants thereof.

[0239] In some embodiments, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO: 2 or SEQ ID NO: 3.

[0240] In some embodiments, the immunoglobulin single variable domain (e.g., VHH) is located at the N-terminus of the immunoglobulin Fc region, and the two are directly connected or connected through a peptide chain, and the peptide chain is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(GGGS) n 、(GGSG) n 、(GGGGS) n and (GAP) n , wherein n is an integer from 1 to 10.

[0241] In some embodiments, the amino acid sequence of the PD-L1 binding molecule is SEQ ID NO: 4.

[0242] In some embodiments, the protein-drug conjugate has the following structure:

[0243] wherein P* is an antibody (the antibody may be as defined above), GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 10;

[0244] Gal* is a modified galactose selected from the following structures:

[0245] LP is:

[0246] k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0247] The core GlcNAc is directly linked to the antibody.

[0248] In some embodiments, k=2, 4, or 8.

[0249] In some embodiments, the modified galactose is linked to the GlcNAc via a β-1,4-glycosidic bond.

[0250] In some embodiments, the core GlcNAc is linked to the Fc fragment of the antibody; 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.

[0251] In some embodiments, the antibody is a bispecific antibody targeting different epitopes of HER2.

[0252] In some embodiments, the antibody has a first heavy chain, a second heavy chain, and a common light chain, wherein the first heavy chain comprises the heavy chain variable region set forth in SEQ ID NO: 17, the second heavy chain comprises the heavy chain variable region set forth in SEQ ID NO: 18, and the common light chain comprises the light chain variable region set forth in SEQ ID NO: 19.

[0253] In some embodiments, the antibody comprises a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 20, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 21, and a common light chain having an amino acid sequence as shown in SEQ ID NO: 22.

[0254] Regarding the content of each component in the composition, the concentration of the protein-drug conjugate in the composition can be 20 mg / mL-150 mg / mL, preferably 50 mg / mL-120 mg / mL, and more preferably 60 mg / mL-100 mg / mL; specifically, the concentration of the protein-drug conjugate can be 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL or 150 mg / mL.

[0255] In some embodiments, the concentration of the antigen binding protein or PD-L1 binding protein in the composition is 20 mg / mL-180 mg / mL, preferably 50 mg / mL-150 mg / mL, more preferably 80 mg / mL-120 mg / mL; specifically, the concentration of the antigen binding protein or PD-L1 binding protein can be 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL. , 70mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 90mg / mL, 95mg / mL, 100mg / mL, 105mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / m L, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL, 150mg / mL, 155mg / mL, 160mg / mL, 165mg / mL, 170mg / mL, 175mg / mL or 180mg / mL.

[0256] In some embodiments, the ratio of protein-drug conjugate to PD-L1 binding molecule in the composition is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0257] In some embodiments, the pH of the composition is 4.0 to 7.0, preferably 4.5 to 6.5, more preferably 5.0 to 6.0; specifically, the pH of the composition can be 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0258] The pH of the composition disclosed herein can be the pH value measured after mixing the protein-drug conjugate, PD-L1 binding molecule and other components, such as the pH value of the composition obtained after liquid replacement, and / or the pH value actually measured after the composition is mixed.

[0259] In some embodiments, the composition of the present invention may contain a buffer component selected from acetate-sodium acetate, histidine-histidine hydrochloride, acetate-histidine, phosphate-sodium phosphate and citric acid-sodium hydroxide; preferably acetate-sodium acetate, histidine-histidine hydrochloride or acetate-histidine.

[0260] In some embodiments, the concentration of the buffer component is 2mM-50mM, preferably 5mM-30mM or 2mM-20mM, more preferably 10mM-20mM or 2mM-10mM; specifically, the concentration of the buffer component can be 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM.

[0261] The buffer component concentration may be the concentration of the replacement fluid and / or the buffer component concentration after the fluid exchange; they may be the same or slightly different.

[0262] In some embodiments, the concentration of the buffer component is the concentration of the buffer component in the replacement fluid.

[0263] In some embodiments, the concentration of the buffer component is the concentration of the buffer component in the composition after fluid exchange.

[0264] In some embodiments, the composition may further include an excipient, wherein the excipient includes a stabilizer, and the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, proline, glycine, arginine hydrochloride and glycerol, preferably selected from one or more of sucrose, trehalose, mannitol, sorbitol and proline, more preferably selected from one or more of trehalose, mannitol, sorbitol and proline, and even more preferably proline.

[0265] In some embodiments, the concentration of the stabilizer is 50mM-250mM, preferably 80mM-200mM, more preferably 100mM-150mM; specifically, the concentration of the stabilizer can be 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 1 mM, 230mM, 235mM, 240mM, 245mM or 250mM.

[0266] In some specific embodiments, the stabilizer selected is proline, trehalose, mannitol or sorbitol, and the concentration of the aforementioned stabilizer is 50mM-250mM, preferably 80mM-200mM, and more preferably 100mM-150mM.

[0267] In some embodiments, the auxiliary material may optionally further comprise an antioxidant, which may be selected from methionine, phenol, cresol, ascorbic acid, glutathione, sodium thiosulfate, citric acid and nicotinamide; preferably methionine.

[0268] In some embodiments, the concentration of the antioxidant is 10mM-100mM, preferably 15mM-75mM, more preferably 20mM-50mM, and even more preferably 30mM-40mM; specifically, the concentration of the antioxidant can be 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM.

[0269] In some embodiments, the composition further comprises a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188. The present application uses mg / mL or % (w / v) to express the concentration of the surfactant. Those skilled in the art will appreciate that the two can be converted. For example, 0.02% surfactant can be converted to 0.2 mg / mL.

[0270] In some embodiments, the concentration of the surfactant is 0 mg / mL-0.8 mg / mL, preferably 0.05 mg / mL-0.75 mg / mL, more preferably 0.1 mg / mL-0.5 mg / mL, even more preferably 0.2 mg / mL-0.4 mg / mL or 0.1 mg / mL-0.3 mg / mL; specifically, the concentration of the surfactant can be 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL or 0.8 mg / mL.

[0271] In some specific embodiments, the composition may comprise a formulation selected from the group consisting of:

[0272] (1) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0273] (2) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM sucrose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0274] (3) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM trehalose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0275] (4) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM mannitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0276] (5) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM sorbitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0277] (6) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0278] (7) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM sucrose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0279] (8) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM trehalose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0280] (9) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM mannitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0281] (10) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM sorbitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0282] (11) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0;

[0283] (12) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 2 mM-20 mM histidine-acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; or

[0284] (13) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 2 mM-20 mM histidine-histidine hydrochloride, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0285] The concentration of the buffer component may be the concentration in the replacement fluid before fluid exchange or the concentration in the composition after fluid exchange.

[0286] In some specific embodiments, the composition may comprise a formulation selected from the group consisting of:

[0287] (a) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 140 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0288] (b) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-acetate, 140 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0289] (c) 100 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0290] (d) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM sucrose, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0291] (e) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM trehalose, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0292] (f) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM mannitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0293] (g) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM sorbitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0294] (h) 60 mg / mL protein-drug conjugate, 90 mg / mL PD-L1 binding protein, 10 mM histidine-histidine hydrochloride, 180 mM sorbitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0295] In some embodiments, the concentration of the buffer component can be the concentration in the replacement fluid before fluid exchange.

[0296] In other specific embodiments, the composition may comprise a formulation selected from the group consisting of:

[0297] (i) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 140 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0298] (j) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-acetate, 140 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0299] (k) 100 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM proline, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0300] (l) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM sucrose, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0301] (m) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM trehalose, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0302] (n) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM mannitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0303] (o) 80 mg / mL protein-drug conjugate, 100 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM sorbitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0304] (p) 60 mg / mL protein-drug conjugate, 90 mg / mL PD-L1 binding protein, 2 mM-5 mM histidine-histidine hydrochloride, 180 mM sorbitol, 30 mM methionine, 0.2 mg / mL polysorbate 80, pH 5.0 to 6.0.

[0305] In some embodiments, the concentration of the buffer component can be the concentration in the composition after liquid exchange.

[0306] In some embodiments, the composition of the present invention has an osmotic pressure suitable for subcutaneous injection, for example, 200 mOsmo / kg to 400 mOsmo / kg, preferably 250 mOsmo / kg to 350 mOsmo / kg, and more preferably 280 mOsmo / kg to 320 mOsmo / kg.

[0307] In some embodiments, the composition of the present invention has a viscosity suitable for subcutaneous injection, for example, 2 cP-25 cP, preferably 5 cP-20 cP, more preferably 8 cP-15 cP.

[0308] In some embodiments, the PD-L1 binding protein in the composition of the invention is KN035 (Envolimab).

[0309] In some embodiments, the protein-drug conjugate in the composition of the present invention is JSKN003.

[0310] In some embodiments, the compositions of the present invention are free of sodium chloride.

[0311] Drug combinations

[0312] 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) a programmed death ligand 1 (PD-L1) binding molecule of the invention and (ii) a protein-drug conjugate of the invention) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. 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 dosages and / or time intervals 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.

[0313] Therefore, in a third aspect, the present application further provides a pharmaceutical combination or combination product comprising the antigen-binding molecule and protein-drug conjugate of the present invention, wherein the definitions of the antigen-binding molecule 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.

[0314] The present application also provides a kit comprising the drug combination, for example, the kit comprises in the same package:

[0315] - a first container containing an antigen-binding molecule of the present invention or a pharmaceutical composition comprising the antigen-binding molecule;

[0316] - a second container containing the protein-drug conjugate of the present invention or a pharmaceutical composition comprising the protein-drug conjugate;

[0317] The limitations on antigen-binding molecules and protein-drug conjugates can refer to the technical features described in the first aspect.

[0318] 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.

[0319] Treatment methods and uses

[0320] In a fourth aspect, the present application also provides a method for treating tumors or cancers, such as advanced or metastatic solid malignancies, comprising administering a drug combination of a programmed death ligand 1 (PD-L1) binding molecule and a protein-drug conjugate to a patient in need.

[0321] In some embodiments, the present application also provides a method for treating a tumor or cancer, such as an advanced or metastatic solid malignant tumor, comprising administering a programmed death ligand 1 (PD-L1) binding molecule combined with a protein-drug conjugate to a patient in need thereof.

[0322] 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.

[0323] In a fifth aspect, the present application also provides a method for treating tumors or cancers, such as advanced or metastatic solid malignant tumors, comprising administering the pharmaceutical composition of the present application to a patient in need.

[0324] The limitations of the PD-L1 binding molecules and protein-drug conjugates in the treatment method can refer to the technical features described in the first aspect.

[0325] In some embodiments, the malignant tumor is an advanced / unresectable or metastatic solid malignant tumor and expresses HER2. HER2 expression is defined as immunohistochemistry (IHC) ≥1+, and HER2 mutations in NSCLC patients are considered HER2 expression.

[0326] In some embodiments, the solid malignancy is a gastrointestinal tumor or non-small cell lung cancer.

[0327] In some embodiments, the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the drug combination is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0328] In some embodiments, the ratio of protein-drug conjugate to PD-L1 binding molecule in the pharmaceutical composition is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0329] In some embodiments, the method of treatment comprises administering the pharmaceutical combination or pharmaceutical composition to a patient in need thereof once a week, once every two weeks, once every three weeks, or once every four weeks.

[0330] In some embodiments, the pharmaceutical combination or pharmaceutical composition is administered by subcutaneous injection or intravenous injection; preferably by subcutaneous injection.

[0331] In some embodiments, the single administration dose of the drug combination can be 0.5 mg / kg-10.0 mg / kg, preferably 0.8 mg / kg-7.5 mg / kg, more preferably 1.0 mg / kg-7.0 mg / kg, based on the patient's body weight, for example, selected from 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg and 6.7 mg / kg, wherein the dose refers to the sum of the doses of the protein-drug conjugate and the PD-L1 binding molecule.

[0332] For example, the aforementioned doses of the drug combination respectively comprise 0.49 mg / kg of protein-drug conjugate and 0.61 mg / kg of PD-L1 binding molecule (dose of 1.1 mg / kg), 1.02 mg / kg of protein-drug conjugate and 1.28 mg / kg of PD-L1 binding molecule (dose of 2.3 mg / kg), 2.0 mg / kg of protein-drug conjugate and 2.5 mg / kg of PD-L1 binding molecule (dose of 4.5 mg / kg), 2.49 mg / kg of protein-drug conjugate and 3.11 mg / kg of PD-L1 binding molecule (dose of 5.6 mg / kg), and 2.98 mg / kg of protein-drug conjugate and 3.72 mg / kg of PD-L1 binding molecule (dose of 6.7 mg / kg).

[0333] In some embodiments, the single administration dose of the pharmaceutical composition can be 0.5 mg / kg-10.0 mg / kg, preferably 0.8 mg / kg-7.5 mg / kg, more preferably 1.0 mg / kg-7.0 mg / kg, based on the patient's body weight, for example, selected from 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg and 6.7 mg / kg, wherein the dose refers to the sum of the doses of the protein-drug conjugate and the PD-L1 binding molecule in the composition.

[0334] For example, the aforementioned doses of the pharmaceutical composition respectively contain 0.49 mg / kg of protein-drug conjugate and 0.61 mg / kg of PD-L1 binding molecule (dose of 1.1 mg / kg), 1.02 mg / kg of protein-drug conjugate and 1.28 mg / kg of PD-L1 binding molecule (dose of 2.3 mg / kg), 2.0 mg / kg of protein-drug conjugate and 2.5 mg / kg of PD-L1 binding molecule (dose of 4.5 mg / kg), 2.49 mg / kg of protein-drug conjugate and 3.11 mg / kg of PD-L1 binding molecule (dose of 5.6 mg / kg), and 2.98 mg / kg of protein-drug conjugate and 3.72 mg / kg of PD-L1 binding molecule (dose of 6.7 mg / kg).

[0335] In some specific embodiments, the treatment method comprises administering the drug combination to a patient in need thereof once weekly by subcutaneous injection, with a single administration dose of 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg, or 6.7 mg / kg. Preferably, in the drug combination, the ratio of the protein-drug conjugate to the PD-L1 binding molecule is 1:1.25.

[0336] In some specific embodiments, the treatment method comprises administering the pharmaceutical composition to a patient in need thereof by subcutaneous injection once a week, with a single dose of 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg, or 6.7 mg / kg. Preferably, in the pharmaceutical composition, the ratio of the protein-drug conjugate to the PD-L1 binding molecule is 1:1.25.

[0337] In a fourth aspect, the present application also relates to a drug combination or pharmaceutical composition of a programmed death ligand 1 (PD-L1) binding molecule and a protein-drug conjugate, which can be used to treat advanced or metastatic solid malignant tumors.

[0338] The limitations of the PD-L1 binding molecules and protein-drug conjugates in the drug combination or pharmaceutical composition can refer to the technical features described in the first aspect.

[0339] In some embodiments, the malignant tumor is an advanced / unresectable or metastatic solid malignant tumor and expresses HER2. HER2 expression is defined as immunohistochemistry (IHC) ≥1+, and HER2 mutations in NSCLC patients are considered HER2 expression. Alternatively, the solid malignant tumor is a gastrointestinal tumor or non-small cell lung cancer.

[0340] In some embodiments, the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the drug combination is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0341] In some embodiments, the ratio of protein-drug conjugate to PD-L1 binding molecule in the pharmaceutical composition is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0342] In some embodiments, the treatment comprises administering the pharmaceutical combination or composition to a patient in need thereof once a week, once every two weeks, once every three weeks, or once every four weeks.

[0343] In some embodiments, the pharmaceutical combination or pharmaceutical composition is administered by subcutaneous injection or intravenous injection; preferably by subcutaneous injection.

[0344] In some embodiments, the single administration dose of the drug combination can be 0.5 mg / kg-10.0 mg / kg, preferably 0.8 mg / kg-7.5 mg / kg, more preferably 1.0 mg / kg-7.0 mg / kg, based on the patient's body weight, for example, selected from 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg and 6.7 mg / kg, wherein the dose refers to the sum of the doses of the protein-drug conjugate and the PD-L1 binding molecule.

[0345] For example, the aforementioned doses of the drug combination respectively comprise 0.49 mg / kg of protein-drug conjugate and 0.61 mg / kg of PD-L1 binding molecule (dose of 1.1 mg / kg), 1.02 mg / kg of protein-drug conjugate and 1.28 mg / kg of PD-L1 binding molecule (dose of 2.3 mg / kg), 2.0 mg / kg of protein-drug conjugate and 2.5 mg / kg of PD-L1 binding molecule (dose of 4.5 mg / kg), 2.49 mg / kg of protein-drug conjugate and 3.11 mg / kg of PD-L1 binding molecule (dose of 5.6 mg / kg), and 2.98 mg / kg of protein-drug conjugate and 3.72 mg / kg of PD-L1 binding molecule (dose of 6.7 mg / kg).

[0346] In some embodiments, the single administration dose of the pharmaceutical composition can be 0.5 mg / kg-10.0 mg / kg, preferably 0.8 mg / kg-7.5 mg / kg, more preferably 1.0 mg / kg-7.0 mg / kg, based on the patient's body weight, for example, selected from 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg and 6.7 mg / kg, wherein the dose refers to the sum of the doses of the protein-drug conjugate and the PD-L1 binding molecule.

[0347] For example, the aforementioned doses of the pharmaceutical composition respectively contain 0.49 mg / kg of protein-drug conjugate and 0.61 mg / kg of PD-L1 binding molecule (dose of 1.1 mg / kg), 1.02 mg / kg of protein-drug conjugate and 1.28 mg / kg of PD-L1 binding molecule (dose of 2.3 mg / kg), 2.0 mg / kg of protein-drug conjugate and 2.5 mg / kg of PD-L1 binding molecule (dose of 4.5 mg / kg), 2.49 mg / kg of protein-drug conjugate and 3.11 mg / kg of PD-L1 binding molecule (dose of 5.6 mg / kg), and 2.98 mg / kg of protein-drug conjugate and 3.72 mg / kg of PD-L1 binding molecule (dose of 6.7 mg / kg).

[0348] In some specific embodiments, the treatment comprises administering the drug combination to a patient in need thereof once weekly by subcutaneous injection, with a single dose of 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg, or 6.7 mg / kg. Preferably, the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the drug combination is 1:1.25.

[0349] In some specific embodiments, the treatment comprises administering the pharmaceutical composition to a patient in need thereof once weekly by subcutaneous injection, with a single dose of 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg, or 6.7 mg / kg. Preferably, the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the pharmaceutical composition is 1:1.25.

[0350] In a fifth aspect, the present invention relates to the use of programmed death ligand 1 (PD-L1) binding molecules combined with protein-drug conjugates in the preparation of drugs for the treatment of advanced or metastatic solid malignant tumors.

[0351] The present invention also relates to the use of programmed death ligand 1 (PD-L1) binding molecules in the preparation of combined protein-drug conjugates for the treatment of advanced or metastatic solid malignant tumors.

[0352] The present invention also relates to the use of protein-drug conjugates in the preparation of drugs for treating advanced or metastatic solid malignant tumors in combination with programmed death ligand 1 (PD-L1) binding molecules.

[0353] The present invention also relates to the use of a drug combination or pharmaceutical composition of a programmed death ligand 1 (PD-L1) binding molecule and a protein-drug conjugate in the preparation of a drug for treating advanced or metastatic solid malignant tumors.

[0354] The limitations of the PD-L1 binding molecules and protein-drug conjugates in the drug combination or pharmaceutical composition can refer to the technical features described in the first aspect.

[0355] In some embodiments, the malignant tumor is an advanced / unresectable or metastatic solid malignant tumor and expresses HER2. HER2 expression is defined as immunohistochemistry (IHC) ≥1+, and HER2 mutations in NSCLC patients are considered HER2 expression. Alternatively, the solid malignant tumor is a gastrointestinal tumor or non-small cell lung cancer.

[0356] In some embodiments, the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the drug combination is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0357] In some embodiments, the ratio of protein-drug conjugate to PD-L1 binding molecule in the pharmaceutical composition is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.25.

[0358] Antigen-binding molecules and protein-drug conjugates

[0359] In a sixth aspect, the antigen-binding molecules and protein-drug conjugates described in aspects 1 to 5 may be further defined as follows:

[0360] In some embodiments, the antigen binding molecule is a programmed death-ligand 1 (PD-L1) binding molecule.

[0361] In some embodiments, the PD-L1 binding molecule comprises an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO: 1. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

[0362] In some embodiments, the CDR1, CDR2, and CDR3 in the VHH set forth in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NOs: 5-7 (Kabat), SEQ ID NOs: 8-10 (AbM), SEQ ID NOs: 11-13 (Chothia), and SEQ ID NOs: 14-16 (IMGT).

[0363] In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 1. Preferably, the amino acid sequence of the immunoglobulin single variable domain is shown in SEQ ID NO: 1.

[0364] Optionally, the aforementioned PD-L1 binding molecule further comprises an immunoglobulin Fc region. Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region, such as the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or variants thereof.

[0365] In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0366] In some embodiments, the immunoglobulin single variable domain can be located at the N-terminus of the immunoglobulin Fc region, or at the C-terminus of the immunoglobulin Fc region; the two are directly connected or connected through a peptide chain.

[0367] In some embodiments, the aforementioned peptide chain is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(GGGS) n 、(GGSG) n 、(GGGGS) n and (GAP) n , wherein n is an integer from 1 to 10.

[0368] In some embodiments, the PD-L1 binding molecule comprises the amino acid sequence shown in SEQ ID NO: 4; preferably, the amino acid sequence of the PD-L1 binding molecule is shown in SEQ ID NO: 4.

[0369] In some embodiments, the protein-drug conjugate has the structure of Formula I:

[0370] P-(LD)n(I), wherein P is a protein, L is a linker unit, D is a substance with biological activity; and n is an integer from 1 to 20.

[0371] In some embodiments, the protein P has desired physiological properties, and preferably, the protein P can prevent and / or treat a disease or condition. Specifically, the protein P can be an antibody, a fusion protein, a cytokine, a polypeptide, and the like.

[0372] In some embodiments, the protein P is an antibody; the antibody targets one or more of the following antigens: 4-1BB, 4-1BBL, A33, adenosine A2a receptor, Akt, ALK, androgen receptor, Ang-1, Ang-2, Annexin A3, Aurora A, Aurora B, B7-H3, B7-H4, Bcl-2, Bcr-Abl, BRAF, BTK, BTLA, BTN2A1, CA-125, CAIX, CCR4, CD105 / endoglin, CD109, CD123, CD155, CD16, CD160, CD19, CD20, CD200, CD200R, CD22, CD24 , CD25, CD27, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD40L, CD47, CD48, CD52, CD70, CD79b, CD80, CD86, CD96, CDK4, CDK6, CDK9, CEA, CEACAM1, ChK1, ChK2, c-KIT, c-Met, C OX2, CSF-1R, CSF2, CTLA-4, CXCR2, CXCR4, DDR2, DLL3, DLL4, DNAM-1, DR5, EGFR, EpCAM, EPHA3, EphB4, ERK1, ERK2 / p38MAPK, FAK, FAP, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, Flt -3, Gal-9, GITR, GITRL, Glypican-3, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HER2, HER3, HER4 / ERBB4, HGF, HHLA2, HIF-1α, HSP27, HSP90, HVEM, ICOS, ICOS Ligand, IDO, IGF1R, IL-13, IL-6, JAK1, JAK2, JAK3, KRAS, LAG-3, LIGHT, MDM2, MEK1, MEK2, MMP-1, MMP-10, MMP-11, MMP-13, MMP-2, MMP-7, MMP-9, mTOR, Mucin1. Myc, NF-κB, NKG2A, NRAS, NTRK1, NTRK2, NTRK3, OX40, OX40L, p53, PAF, PARP1, PARP2, PD1, PDGFR-α, PDGFR-β, PD-L1, PD-L2, PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ, PIM1 , PIM3, PSMA, PTEN, RAF-1, RANKL, RET, S100A4, SIRPα, SLAMF7, SMO, Src, STAT3, STEAP-1, SYK, TDO, TGFβ, Tie-2, TIGIT, TIM-3, TLR8, TMIGD2, TNF-α, Toll-like receptor 3. TRAIL, TRAILR1, TROP-2, VEGF, VEGF-C, VEGFR-1, VEGFR-2, VEGFR-3 and VISTA.

[0373] In some embodiments, the antibody can target HER2; preferably, the antibody can bind to different epitopes of human HER2, such as the extracellular domain II of human HER2, and / or the extracellular domain IV of human HER2;

[0374] In some embodiments, the antibody is a bispecific antibody targeting different epitopes of HER2. In some embodiments, the antibody can specifically bind to extracellular domain II and extracellular domain IV of human HER2.

[0375] In some embodiments, the antibody comprises a first heavy chain, a second heavy chain, and a common light chain, wherein the first heavy chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 17, the second heavy chain comprises CDR1, CDR2, and CDR3 of the heavy chain variable region set forth in SEQ ID NO: 18, and the common light chain comprises CDR1, CDR2, and CDR3 of the light chain variable region set forth in SEQ ID NO: 19. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.

[0376] Wherein, CDR1, CDR2 and CDR3 in the heavy chain variable region represented by SEQ ID NO: 17 are selected from any one of the following groups: SEQ ID NO: 23-25 ​​(Kabat), SEQ ID NO: 26-28 (AbM), SEQ ID NO: 29-31 (Chothia) and SEQ ID NO: 32-34 (IMGT).

[0377] The CDR1, CDR2 and CDR3 in the heavy chain variable region represented by SEQ ID NO: 18 are selected from any one of the following groups: SEQ ID NO: 35-37 (Kabat), SEQ ID NO: 38-40 (AbM), SEQ ID NO: 41-43 (Chothia) and SEQ ID NO: 44-46 (IMGT).

[0378] The CDR1, CDR2 and CDR3 in the light chain variable region represented by SEQ ID NO: 19 are selected from any one of the following groups: SEQ ID NO: 47-49 (Kabat), SEQ ID NO: 50-52 (AbM), SEQ ID NO: 53-55 (Chothia) and SEQ ID NO: 56-58 (IMGT).

[0379] In some embodiments, the antibody comprises the first heavy chain variable region set forth in SEQ ID NO:17.

[0380] In some embodiments, the antibody comprises a second heavy chain variable region set forth in SEQ ID NO:18.

[0381] In some embodiments, the antibody comprises the common light chain variable region set forth in SEQ ID NO:19.

[0382] In some embodiments, the amino acid sequence of the first heavy chain of the antibody is shown in SEQ ID NO:20.

[0383] In some embodiments, the amino acid sequence of the second heavy chain of the antibody is as shown in SEQ ID NO:21.

[0384] In some embodiments, the amino acid sequence of the common light chain of the antibodies is shown in SEQ ID NO:22.

[0385] In some embodiments, the linker unit L in Formula I has a structure shown in Formula II:

[0386] -L1-sp1-L2-sp2-(II)

[0387] Among them, L1 is a linker for connecting to P, sp1 is the first spacer unit, L2 is a cleavable linker, and sp2 is the second spacer unit and is connected to D.

[0388] In some embodiments, the linker unit L in Formula I has a structure shown in Formula II:

[0389] -L1-sp1-L2-sp2-(II)

[0390] wherein L1 is a linker for connecting to P, sp1 is a first spacer unit, L2 is absent, and sp2 is a second spacer unit and is connected to D. In some embodiments, L1 in Formula II is selected from:

[0391] (The side connected to the protein is marked as P, and the side connected to the first spacer unit is marked as sp1),

[0392] Among them, 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.

[0393] In some embodiments, L1 is

[0394] In some embodiments, the structure of the first spacer unit sp1 in Formula II is:

[0395] (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.

[0396] In some embodiments, in the structure of sp1, a1=1; in other embodiments, a1=0.

[0397] In some embodiments, a2=0, 1, 2, 3, 4, 5, or 6.

[0398] In some embodiments, b1=0 or 1.

[0399] In some embodiments, b2=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0400] In some embodiments, b3=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0401] In some embodiments, c=0, 1, 2, 3, 4, 5, or 6.

[0402] 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.

[0403] In some specific embodiments, the structure of sp1 is selected from the following group:

[0404] (1) a1=0, a2=2, 3, 4, 5 or 6, b1=0, b2=0, b3=0, c=0;

[0405] (2) a1=0, a2=0, b1=0, b2=0, b3=0, c=2, 3, 4, 5, or 6;

[0406] (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;

[0407] (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

[0408] (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;

[0409] In some embodiments, the structure of sp1 is specifically selected from the following group:

[0410] (1.1) a1=0, a2=2, b1=0, b2=0, b3=0, c=0;

[0411] (1.2) a1=0, a2=3, b1=0, b2=0, b3=0, c=0;

[0412] (1.3) a1=0, a2=4, b1=0, b2=0, b3=0, c=0;

[0413] (1.4) a1=0, a2=5, b1=0, b2=0, b3=0, c=0;

[0414] (1.5) a1=0, a2=6, b1=0, b2=0, b3=0, c=0;

[0415] (2.1) a1=0, a2=0, b1=0, b2=0, b3=0, c=2;

[0416] (2.2) a1=0, a2=0, b1=0, b2=0, b3=0, c=3;

[0417] (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">

[0418] <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">

[0419] <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">

[0420] <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">

[0421] <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">

[0422] <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">

[0423] <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">

[0424] <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">

[0425] <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">

[0426] <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">

[0427] <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">

[0428] <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">

[0429] <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">

[0430] <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">

[0431] <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">

[0432] <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">

[0433] <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">

[0434] <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">

[0435] <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">

[0436] <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">

[0437] <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">

[0438] <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">

[0439] <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">

[0440] <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">

[0441] <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">

[0442] <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">

[0443] <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">

[0444] <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">

[0445] <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">

[0446] <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">

[0447] <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">

[0448] <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">

[0449] <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">

[0450] <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">

[0451] <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">

[0452] <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">

[0453] <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">

[0454] <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">

[0455] <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">

[0456] <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">

[0457] <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">

[0458] <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">

[0459] <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">

[0460] <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">

[0461] <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">

[0462] <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">

[0463] <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">

[0464] <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">

[0465] <h2 style=";text-align:left;direction:ltr"> (5.4)a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 5, c = 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0466] (5.5) a1=1, a2=0, b1=0, b2=0, b3=6, c=2;

[0467] (5.6) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 7, c = 2; and

[0468] (5.7) a1=1, a2=0, b1=0, b2=0, b3=8, c=2.

[0469] In some embodiments, the cleavable linker L2 in Formula II is a dipeptide, tripeptide, or tetrapeptide residue.

[0470] 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-.

[0471] 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.

[0472] 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.

[0473] In some embodiments, sp2 in Formula II is absent, or sp2 is selected from:

[0474] (The side connected to L2 is marked as L2, and the side connected to the biologically active substance D is marked as D), wherein,

[0475] 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 C1-6 alkyl;

[0476] The alkyl group may be optionally substituted with hydroxy, amino, halogen, nitro and cyano.

[0477] In some embodiments, sp2 is

[0478] In some embodiments, sp2 is

[0479] 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.

[0480] In some embodiments, the linking unit L can be selected from the following structures:

[0481] k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10. (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).

[0482] In some embodiments, k=2, 4, or 8.

[0483] The biologically active substance D in formula I can be selected from cytotoxins, protein kinase inhibitors, immune agonists, glucocorticoids, oligonucleotides, radioisotopes, polypeptides and any combination thereof.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] In some embodiments, D has the structure shown in Formula III:

[0489] wherein X is selected from CH2, NH, O, S or SO2;

[0490] Y does not exist, or Y has The structure shown;

[0491] wherein W1 and W3 are each independently selected from O, S and NH, and W2 is selected from C, CH and N,

[0492] 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;

[0493] 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;

[0494] 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;

[0495] d and e are each independently selected from integers from 0 to 5.

[0496] In some embodiments, D has the structure shown in Formula III-a:

[0497] Where W1 is O or NH;

[0498] R a 、R b are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 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;

[0499] 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;

[0500] d is an integer from 0 to 5.

[0501] In some embodiments, W1 is O.

[0502] In some embodiments, d is 0, 1, or 2.

[0503] In some embodiments, wherein R a Selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 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.

[0504] In some embodiments, wherein R a and R bTogether 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.

[0505] In some embodiments, D can be selected from the group consisting of:

[0506] In other embodiments, D has the structure shown in Formula III-b:

[0507] Where W3 is O or NH, W2 is C, CH or N,

[0508] 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;

[0509] 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;

[0510] e is an integer from 0 to 5.

[0511] In some embodiments, W3 is O; in other embodiments, W2 is CH.

[0512] In some embodiments, e is 0, 1, or 2.

[0513] 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.

[0514] In some embodiments, D can be selected from the group consisting of:

[0515] In some embodiments, D, as a camptothecin derivative, can also be selected from the following structures:

[0516] (i.e. Exatecan), (i.e. SN-38).

[0517] In some embodiments, D may be an auristatin derivative, preferably selected from the following structures:

[0518] In some embodiments, D may be a maytansine derivative, preferably selected from the following structures:

[0519] In some embodiments, D is eribulin and its derivatives, preferably selected from the following structures:

[0520] In some embodiments, in Formula I, the linker L is connected to the antibody via a thiol group; preferably, the thiol group is derived from the 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.

[0521] In other embodiments, the linker unit L is linked to the antibody via an oligosaccharide; preferably, the oligosaccharide is derived from the natural sugar chain of the antibody.

[0522] In some embodiments, the oligosaccharide is derived from an N-glycan chain of an antibody.

[0523] In some embodiments, the oligosaccharide consists of 2 to 15 monosaccharides; preferably, the oligosaccharide consists of 2 to 10 monosaccharides.

[0524] In some embodiments, the oligosaccharide has the structure shown in Formula Va or Formula Vb:

[0525] wherein P* is an antibody (the antibody may be as defined above), GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 20;

[0526] Gal* is a modified galactose selected from the following structures:

[0527] The oligosaccharide is linked to the antibody via a core GlcNAc.

[0528] In some embodiments, the modified galactose is linked to the GlcNAc via a β-1,4-glycosidic bond.

[0529] In some embodiments, the oligosaccharide is linked to the Fc fragment of the antibody; 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.

[0530] As mentioned above, the oligosaccharide can be derived from the natural sugar chain of the antibody. Specifically, the method for preparing the oligosaccharide precursor comprises the following steps: reacting an antibody whose N-sugar glycoform is mainly G0F with UDP-GalNAz or a salt thereof in the presence of a catalyst to obtain the aforementioned oligosaccharide precursor.

[0531] Methods for obtaining antibodies with G0F glycoforms are well known in the art. For example, antibodies expressed in eukaryotic cells are post-translationally modified and the sugar chains can be converted to G0F form by treatment with β-galactosidase, which removes any terminal galactose residues and leaves a terminal N-acetylglucosamine residue.

[0532] The precursor of the oligosaccharide has the structure shown in Formula Vc:

[0533] Wherein, P*, GlcNAc, Fuc, Man, f and m are as defined above, and Gal*-N3 has the following structure:

[0534] The UDP-GalNAz has the following structure:

[0535] In some embodiments, the aforementioned catalyst is galactosyltransferase or a functional variant or fragment thereof.

[0536] In some embodiments, the catalyst is β-1,4-galactosyltransferase or a functional variant or fragment thereof.

[0537] In some embodiments, the catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase, or a functional variant or fragment thereof.

[0538] 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.

[0539] In some embodiments, the catalyst is β-1,4-acetylgalactosyltransferase disclosed in patent application WO2016170186.

[0540] In some embodiments, the catalyst comprises the sequence set forth in any one of SEQ ID NOs: 59-61.

[0541] In some specific embodiments, the protein-drug conjugate described herein has a structure shown in Formula VI:

[0542] wherein P* is an antibody (the antibody may be as defined above), GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 10;

[0543] Gal* is a modified galactose selected from the following structures:

[0544] LP is selected from one of the following structures (a) to (g):

[0545] (a) and / or

[0546] (b) and / or

[0547] (c) and / or

[0548] (d) and / or

[0549] (e) and / or

[0550] (f) and / or as well as

[0551] (g) and / or k is an integer from 1 to 20, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0552] Among them, the core GlcNAc is directly linked to the antibody.

[0553] In some embodiments, k=2, 4, or 8.

[0554] In some embodiments, m is 1 to 4; in some embodiments, the DAR value of the conjugate as a whole is 1 to 8.

[0555] In some embodiments, m is about 1.2 to 3.5, for example, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5.

[0556] In some embodiments, the DAR value of the conjugate as a whole is about 1.5 to 8, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8. .4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0557] In some embodiments, the antibody comprises a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 20, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 21, and a common light chain having an amino acid sequence as shown in SEQ ID NO: 22.

[0558] It should be understood that the technical solution obtained by any combination of any technical features recorded in the sixth aspect above and any technical features of the technical solutions recorded in the first to fifth aspects is also included in this application. Example

[0559] The present invention is further described below with reference to examples, which should not be construed as limiting the present invention.

[0560] Terms and abbreviations

[0561] Tm: Melting Temperature

[0562] Tagg: Aggregation Temperature

[0563] kD:Diffusion Interaction Parameter

[0564] rH:Hydrated Radius, hydration radius

[0565] PDI: Polymer Dispersity Index, polydispersity index

[0566] Turbidity

[0567] DSF: Differential Scanning Fluorimetry

[0568] DLS: Dynamic Light Scattering

[0569] SEC-HPLC: Size Exclusion High Performance Liquid Chromatography, nrCE-SDS: non-reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate

[0570] CEX-HPLC: Cation Exchange High Performance Liquid Chromatography, cation exchange high performance liquid chromatography RH: Relative Humidity, relative humidity

[0571] Example 1. Preparation of KN035 and JSKN003

[0572] 1.1 Preparation of KN035

[0573] KN035 is an anti-PD-L1 single-domain antibody-Fc fusion protein screened from a camel immune library. It is a homodimer formed by two identical peptide chains, the amino acid sequence of which is shown in SEQ ID NO: 4.

[0574] A vector containing a nucleic acid sequence encoding a fusion protein is transferred into cells, and the corresponding fusion protein is expressed and purified. The specific preparation process can be found in patent application document WO2017020801.

[0575] 1.2 Preparation of JSKN003

[0576] JSKN003 is an antibody-drug conjugate consisting of a bispecific antibody targeting HER2 (hereinafter referred to as antibody A) and a linker-toxin.

[0577] 1.2.1 Preparation of Antibody A

[0578] Antibody A comprises a first heavy chain, a second heavy chain and a common light chain, wherein the amino acid sequence of the first heavy chain is shown in SEQ ID NO: 20, the amino acid sequence of the second heavy chain is shown in SEQ ID NO: 21, and the amino acid sequence of the common light chain is shown in SEQ ID NO: 22.

[0579] A vector containing nucleic acid sequences encoding the first heavy chain, the second heavy chain, and the common light chain is transferred into cells for expression and purification to obtain antibody A. The specific preparation process can be found in patent application document WO2016110267.

[0580] 1.2.2 Preparation of Linker-Toxin

[0581] The following linker-toxin (LP1) was prepared according to the process described in patent application CN113264983A:

[0582] After detection, LP1MS m / z (ESI): 1375.77, and the MS spectrum is shown in Figure 4.

[0583] 1.2.3 Conjugation of Antibody A and Linker-Toxin

[0584] Step 1: Synthesis of Antibody A-(N3)4: Desalt the stock solution of Antibody A (primarily G0F) into HEPES buffer for later use. Ultrapure water, MnCl2 (10 mM), UDP-GalNAz (50 mM), Tris-HCl (10 mM), Antibody A (10 mg / mL), and GalT1 (0.125 mg / mL, SEQ ID NO: 59) were added to a centrifuge tube and reacted at 400 rpm for 12 hours. After the reaction was complete, Protein A was purified, concentrated by ultrafiltration, and desalted into DPBS to obtain Antibody A-(N3)4 (148,796 Da). The reaction equation and UDP-GalNAz structure for this step are shown in Figure 5 , and the mass spectrum of the product is shown in Figure 6 .

[0585] Step 2: Synthesis of the Antibody-Drug Conjugate: LP1 (950 μM), DMSO, antibody A-(N3)4 (63 μM), and ultrapure water were added to a centrifuge tube and reacted at 400 rpm for 12 hours. After the reaction was complete, the product was desalted in DPBS to obtain JSKN003 (DAR = 4, 154,301 Da). The reaction equation is shown in Figure 7, and the mass spectrum of the product is shown in Figure 8.

[0586] Example 2: Stability Study of Mixed Formulation of JSKN003 and KN035

[0587] (1)

[0588] FS0-1: A JSKN003 sample (30.75 mg / ml JSKN003, 20 mM histidine-acetate, pH 5.0) was concentrated by ultrafiltration to 80 mg / ml.

[0589] FS0-2: A JSKN003 sample (30.75 mg / ml JSKN003, 20 mM histidine-acetate, pH 5.0) was concentrated by ultrafiltration to 150 mg / ml and mixed with a KN035 sample (200 mg / ml KN035, 20 mM sodium acetate-acetic acid, 220 mM proline, 0.02% PS20, pH 6.0) in a 1:1 ratio.

[0590] Table 1 Preparation prescription

[0591] The test results show that when JSKN003 exists alone, the insoluble particles and aggregates increase significantly after being placed at 40°C for 10 days; when KN035 exists, the increase of total insoluble particles and aggregates is significantly slowed down under the same test conditions (Table 2, Figures 1 and 2).

[0592] Table 2 Test results of mixed preparations

[0593] (2)

[0594] JSKN003 and KN035 samples were mixed at the desired concentrations, exchanged using a dialysis cartridge, concentrated using an ultrafiltration tube, and then adjusted to the formulation shown in Table 3 by adding a buffer system. After preparation, samples of each formulation were placed in a constant temperature and humidity chamber (40±2°C, 70% RH) for 4 weeks. The insoluble particulate matter of each formulation was tested, and the results are shown in Figure 9.

[0595] The experimental results show that in the histidine-histidine hydrochloride buffer system, when high concentration of JSKN003 exists alone, the insoluble particles increase significantly after being placed at 40°C for 4 weeks, while when KN035 is present, the increase of insoluble particles is significantly slowed down; in the sodium acetate-acetic acid buffer system, when KN035 is present, the increase of insoluble particles also tends to slow down.

[0596] Table 3 Prescription of the mixture of JSKN003 and KN035

[0597] Example 3: Prescription Study of Mixed Formulation of JSKN003 and KN035

[0598] JSKN003 and KN035 samples were mixed at the expected concentration, the liquid was replaced with a dialysis cartridge, concentrated with an ultrafiltration tube, and stabilizers and surfactants were added to adjust to the final required formulation.

[0599] 3.1. First round of prescription screening

[0600] The first round of formulation screening aimed to identify suitable pH, buffer systems, and ionic strengths. JSKN003 and KN035 samples were mixed and then substituted into the formulations listed in Table 4. Colloidal stability and high-temperature stability (40±2°C, RH 70±5%, 11 days) were evaluated. Appearance, insoluble particulate matter, SEC-HPLC, nrCE-SDS, and relative binding activity (HER2) were also tested.

[0601] Table 4 The first round of prescription screening plan

[0602] *: The measured pH value of the sample after the liquid is replaced, the same below.

[0603] 3.1.1FS1-Colloidal Stability

[0604] Samples from different formulations were serially diluted to concentrations of 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, 10 mg / ml, and 20 mg / ml, respectively. The kD values ​​were measured using the Panta DLS module. A kD > 0 indicates a protein tendency toward repulsion and good colloidal stability, while a kD < 0 indicates a protein tendency toward aggregation. The test results are shown in Table 5. The results show that higher NaCl concentrations decrease the kD value; kD values ​​< 0 for FS1-5-L and FS1-5-M indicate poor colloidal stability for high-pH formulations.

[0605] Table 5 FS1-colloidal stability test results

[0606] 3.1.2FS1-High Temperature Stability

[0607] Each formulation sample was placed in a constant temperature and humidity chamber (40±2°C, 70% RH) and tested for appearance, insoluble particles, rH and PDI, SEC-HPLC, and nrCE-SDS. The test results are shown in Table 6 and Figures 10A-10C.

[0608] Table 6 FS1-High temperature stability test results

[0609] 3.1.3 Conclusion of the first round of screening

[0610] All formulations showed normal appearance, good insoluble particles, and no significant change in relative binding activity. Addition of NaCl increased opalescence and high aggregate content. As pH increased, aggregate content increased by SEC-HPLC, while nrCE-SDS fragment content decreased. FS1-3 exhibited the lowest aggregate content and acceptable fragment content. Therefore, FS1-3 (histidine / histidine hydrochloride, pH 6.0) was selected for the next round of formulation screening.

[0611] 3.2. Second round of prescription screening

[0612] The second round of formulation screening aimed to identify suitable stabilizers. JSKN003 and KN035 samples were mixed and then substituted into the formulations listed in Table 7. Viscosity and high-temperature stability (40±2°C, 70% RH, 10 days) were evaluated. Appearance, insoluble particulate matter, SEC-HPLC, nrCE-SDS, CEX-HPLC, and relative binding activity (HER2) were also tested.

[0613] Table 7 Second round of prescription screening plan

[0614] The test results are shown in Table 8 and Figures 11A-11F. After high-temperature storage, FS2-5 showed the lowest aggregate content by SEC-HPLC and the highest main peak content by CEX-HPLC. The appearance, viscosity, and relative binding activity of all formulations were normal. Therefore, FS2-5, containing proline and methionine, was selected for the third round of formulation screening.

[0615] Table 8 FS2-High temperature stability test results

[0616] 3.3. The third round of prescription screening

[0617] The third round of prescription screening aimed to determine pH, protein content, methionine content, and polysorbate 80 content. JSKN003 and KN035 samples were substituted into the respective prescriptions in Table 9. After sample preparation, viscosity was tested, and freeze-thaw, shaking, light exposure, and high-temperature stability were performed. See Table 10 for details. After each stability test, the sample appearance, insoluble particles, rH and PDI, SEC-HPLC, nrCE-SDS, CEX-HPLC, Binding (HER2 and PD-L1), and Free-Drug were tested. The test results of each stability test are shown in Tables 11-15.

[0618] Table 9 The third round of prescription screening plan

[0619] *: The pH value of the sample obtained after the medium change was 6.0, and the pH of the replacement medium before the medium change was 5.5; **: The pH value of the sample obtained after the medium change was 5.5, and the pH of the replacement medium before the medium change was 5.0

[0620] Table 10 Stability plan for the third round of prescription screening

[0621] Table 11 FS3-viscosity test results

[0622] Table 12 FS3-freeze-thaw stability test results

[0623] Table 13 FS3-shaking stability test results

[0624] Table 14 FS3-Light stability test results

[0625] Table 15 FS3-High temperature stability test results

[0626] The results of various stability tests for each test item are summarized in Figures 12A-12E. Studies have shown that FS3-6 exhibits low aggregate content by both high-temperature and freeze-thaw SEC-HPLC, so a pH of 5.5 is preferred. Illumination significantly increases the number of FS3-1 particles, so a methionine content of 30 mM is preferred. No advantage was observed with 0.04% polysorbate 80, so 0.02% polysorbate 80 was selected.

[0627] Based on the above results, the preferred formulation is FS3-6: 80 mg / ml JSKN003, 100 mg / ml KN035, pH 5.5, 10 mM histidine / histidine hydrochloride (the amount in the replacement fluid; the actual histidine content decreases after fluid replacement, see below), 180 mM proline, 30 mM methionine, and 0.02% polysorbate 80. It exhibits excellent high-temperature, freeze-thaw, and shaking stability, and a viscosity of <10 cP, making it suitable for subcutaneous injection.

[0628] Considering that the appropriate osmotic pressure for subcutaneous injection should be approximately 300 mOsmo / kg, the proline concentration was calculated to be adjusted to 140 mM. The final preferred mixed formulation (i.e., JSKN033) is formulated as 80 mg / ml JSKN003, 100 mg / ml KN035, with a pH of 5.5 ± 0.5, 10 mM histidine / histidine hydrochloride, 140 mM proline, 30 mM methionine, and 0.02% polysorbate 80. Alternatively, the buffer system can be prepared using histidine plus glacial acetic acid to adjust the pH.

[0629] 3.4. Confirmation of excipient content

[0630] Samples containing 3-30 mM histidine / acetic acid and 140 mM proline were prepared for stability testing. The test results are shown in Table 16 and Figure 13. The results showed that the samples containing 15 mM and 30 mM histidine exhibited increased opalescence and insoluble particles, while no differences were observed in other test parameters. Therefore, the optimal histidine content was determined to be 3-5 mM (with a replacement solution of approximately 10 mM), the proline content to be 140-180 mM, and the buffer system to be either histidine / histidine hydrochloride or histidine / acetate.

[0631] Table 16 Appearance test results for confirmation of histidine content

[0632] *: The replacement buffer was 10 mM and the pH was adjusted with acetic acid. The measured histidine content of the sample after the replacement was approximately 3.3 mM.

[0633] **: After replacing the His 3mM buffer, add buffer to the samples to adjust the histidine content to the target value.

[0634] 3.5 Long-term stability and accelerated stability of mixed preparations

[0635] Scaled-up production was performed according to the final formulation: 10 mM histidine / acetic acid (the replacement solution contained 10 mM histidine, and the measured histidine content after the replacement solution was approximately 4.3 mM), pH 5.5 (the replacement solution had a pH of 5.0, and the pH after the replacement solution was 5.5), 140 mM proline, 30 mM methionine, and 0.02% polysorbate 80. Long-term stability (2-8°C, inverted, 12 M) and accelerated stability (25±2°C, RH 60±5%, inverted, 6 M) were shown in Figures 14A-14D and 15A-15D, demonstrating good long-term and accelerated stability of the mixed formulation.

[0636] Example 4: Subcutaneous administration of mixed preparations

[0637] 4.1 Pharmacokinetic Study of Single Intravenous or Subcutaneous Administration in Female Bama Minipigs

[0638] Ten female Bama miniature pigs were selected and divided into two groups of five. Each group received an intravenous injection of JSKN003 or a subcutaneous injection of JSKN033 according to the following regimen. Blood samples were collected at specific times:

[0639] The drug concentration of JSKN003 in the serum samples was measured, and the results are shown in Figure 3. Following subcutaneous injection of 11.6 mg / kg JSKN003 (equivalent to 5 mg / kg JSKN003), JSKN003 in the compound was rapidly absorbed, reaching maximum plasma concentrations 24-48 hours after injection. Mean Cmax and AUC(0-last) values ​​were 30,420 ng / mL and 7,499,765 ng.h / mL, respectively. The half-life of subcutaneous injection was 164 hours, similar to that of intravenous bolus injection. The mean bioavailability of JSKN0033 after subcutaneous administration was 73.4%.

[0640] 4.2. Intermittent Subcutaneous Injection Stimulation Study in Rabbits

[0641] Eight rabbits were randomly assigned to two groups of two males and two females in each group. The potential for local injection site irritation of JSKN033 was determined by comparing irritation responses on different sides of the dorsal region of the same animal when the test article (left side) or control article (right side) was administered subcutaneously once weekly (days 1 and 8). The test article was injected into the left side of the dorsal region at varying doses (0.25 and 0.5 mL / site), while the control article (sodium chloride injection) was injected into the right side of the dorsal region. The dose concentration was 186.1 mg / mL. At the start of dosing, male and female rabbits were approximately 5 months old, with body weights ranging from 3.2 to 3.4 kg for males and 3.1 to 3.3 kg for females. Necropsies were performed on days 11 (one animal / sex / group) and 15 (an additional animal / sex / group). Evaluation criteria included mortality, clinical signs, body weight, injection site irritation score, and macroscopic and histopathological examinations.

[0642] Results showed no unexpected deaths, clinical signs or weight changes related to the test article, and no macroscopic or histopathological changes were observed at the end of the dosing and recovery periods.

[0643] The daily mean score for the dosing site during both the dosing and recovery phases was 0 for Control (0.25 mL / site), Control (0.5 mL / site), JSKN033 (0.25 mL / site), and JSKN033 (0.5 mL / site).

[0644] The primary irritation index (PII) was derived from the results of the daily average dosing site scores during the dosing phase and the recovery phase. The results showed that during the dosing phase and the recovery phase, the PII for the dosing sites of the control (0.25 mL / site), control (0.5 mL / site), JSKN033 (0.25 mL / site), and JSKN033 (0.5 mL / site) were all 0, indicating that no irritation was observed at the dosing site in all treatment groups.

[0645] Example 5. Phase I / II clinical study evaluating the safety, tolerability, pharmacokinetics / pharmacodynamics, and efficacy of JSKN033 in patients with advanced or metastatic solid malignancies

[0646] Study objectives and endpoints

[0647] Primary objectives and endpoints:

[0648] Secondary objectives and endpoints:

[0649] Study population

[0650] The study will enroll patients with advanced unresectable or metastatic solid malignancies expressing HER2 who are intolerant to standard therapy or have relapsed during or after prior standard systemic therapy, including but not limited to gastric, colorectal, NSCLC, and other solid malignancies. HER2 expression is defined as immunohistochemistry (IHC) ≥1+. HER2 mutations in NSCLC patients are considered HER2-expressing. For the dose expansion phase, only patients with gastrointestinal tumors that express HER2 will be enrolled.

[0651] Sample size

[0652] Dose escalation phase (Phase I): The sample size was determined by the "3+3" design.

[0653] Dose escalation phase (Phase II): SMC will select 1-2 dose cohorts for expansion, adding 10 to 30 patients to each cohort to further explore the safety and efficacy of JSKN033.

[0654] Study Drug

[0655] JSKN033 is available in 180mg / 1mL vial, containing 80mg of JSKN003 and 100mg of Envolizumab. JSKN033 is administered by subcutaneous injection weekly on Day 1, starting with 1.1mg / kg, followed by 2.3mg / kg, 4.5mg / kg, 5.6mg / kg, and 6.7mg / kg.

[0656] Study Design

[0657] This study is an open-label, multicenter, first-in-human Phase I / II (dose escalation and dose expansion) study designed to evaluate the safety, tolerability, PK, immunogenicity, and efficacy of JSKN033 in patients with advanced unresectable or metastatic solid malignancies predicted to express HER2 (IHC ≥1+).

[0658] Dose escalation phase

[0659] The planned dose escalation schedule is as follows:

[0660] The dose escalation phase will use single-patient accelerated dose titration (ADT) for dose level 1 (1.1 mg / kg, SC, QW) and dose level 2 (2.3 mg / kg, SC, QW). Dose level 3 (4.5 mg / kg, SC, QW), dose level 4 (5.6 mg / kg, SC, QW), and dose level 5 (6.7 mg / kg, SC, QW) will be enrolled and monitored using a "3+3" design to determine the MTD / RP2D of JSKN033. If deemed necessary, the Safety Monitoring Committee (SMC) has the right to decide to escalate the dose to other doses (higher, intermediate, or lower doses, or other dose intervals).

[0661] Enrolled patients will be sequentially assigned to the planned dose level according to the protocol and receive JSKN033SC QW treatment to observe the occurrence of treatment-related AEs and dose-limiting toxicities (DLTs). The DLT observation period is 21 days from the first dose of JSKN033.

[0662] Accelerated Dose Titration Dose Escalation: To minimize potential underexposure in the early stages of dose escalation, the study will enroll only one patient at both dose level 1 and dose level 2. If these two patients do not experience a DLT, subsequent patients will be enrolled in dose level 3 of the "3+3" design. If a patient experiences a DLT at dose level 1 or 2, the SMC will decide whether to enroll additional patients at that dose level to assess the safety of JSKN033. If dose level 2 is confirmed to be safe, patients at dose level 1 can be upgraded to dose level 2. If dose level 3 is confirmed to be safe, patients at both dose levels 1 and 2 can be upgraded to dose level 3.

[0663] If a patient receives less than 80% of the planned dose during the DLT observation period, an additional patient will be enrolled. If a patient is unable to receive an adequate dose of JSKN033 due to an adverse event, eligibility for the DLT evaluation will be determined by the SMC.

[0664] Dose expansion phase (Phase II)

[0665] Following or during dose escalation, the SMC will select 1-2 dose levels for expansion, enrolling an additional 10-30 patients with HER2-expressing gastrointestinal tumors per dose level to further explore the efficacy and safety of JSKN033.

[0666] Inclusion criteria

[0667] Patients must meet the following inclusion criteria to participate in the study:

[0668] 1. Willing and able to provide written informed consent (ICF) for the trial.

[0669] 2. Male or female, 18 years of age or older; willing and able to complete all required research procedures.

[0670] 3. Eastern Cooperative Oncology Group performance status 0 or 1, life expectancy ≥ 12 weeks.

[0671] 4. Patients must have pathologically documented advanced / unresectable or metastatic solid malignancies (gastrointestinal tumors in the dose expansion phase) with HER2 expression (IHC ≥ 1+), and be refractory or intolerant to standard treatment, or have no effective standard treatment. HER2 mutations in NSCLC patients are also considered HER2-expressing.

[0672] 5. Measurable disease at baseline according to RECIST 1.1. Target lesions located in previously irradiated areas are considered measurable if progression has been demonstrated in such lesions.

[0673] 6. Adequate organ function assessed within 7 days before the first trial [no blood transfusion, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) or other relevant medical support within 14 days before administration of the study product]:

[0674] 7. Adequate treatment washout period before the first dose, defined as:

[0675] 8. LVEF ≥ 50% as measured by echocardiography (ECHO) or multi-gated acquisition (MUGA) within 28 days before the first dose.

[0676] 9. Female or male patients of childbearing potential agree to use highly effective contraceptive measures (annual failure rate less than 1%) from the first dose to 180 days after the end of the dose. Female subjects of childbearing potential must have a negative pregnancy test result within 7 days before the first dose (fertile potential is defined as premenopausal women without a record of tubal ligation or hysterectomy, or women within 1 year of menopause).

[0677] result

[0678] As of August 20, 2024, a total of ten patients were enrolled (n=4 breast cancer, n=2 lung cancer, n=2 biliary tract cancer, n=1 colorectal cancer, and n=1 salivary gland cancer). Their HER2 expression / mutation status is shown in the table below. Patients received JSKN033 at doses of 1.1 mg / kg (n=1), 2.3 mg / kg (n=1), 4.5 mg / kg (n=3), 5.6 mg / kg (n=3), and 6.7 mg / kg (n=2). The most common treatment-related adverse events (TRAEs) were mild to moderate injection site reactions (grade 1-2). No grade 3 or higher TRAEs or serious adverse events were observed, and no TRAEs led to treatment discontinuation.

[0679] Eight patients were evaluable for efficacy, two of whom showed partial responses and four showed stable disease, for a disease control rate of 75%. Two patients treated with 5.6 mg / kg showed partial responses on their first post-baseline scan. One of these patients had HR-positive / HER2-low-expressing (IHC 2+, FISH-) breast cancer and had received at least four lines of therapy, and the other had HER2-mutated non-small cell lung cancer that had progressed after immunotherapy, chemotherapy, and anti-HER2 therapy.

[0680] In this first-in-human trial of JSKN033, the most common treatment-related adverse events (TRAEs) were mild to moderate injection site reactions, demonstrating its good safety profile. In the 5.6 mg / kg dose group, two patients who had received extensive prior treatment achieved partial responses, which was consistent with estimates from clinical pharmacology data. These results encourage further clinical development of JSKN033. In addition, this formulation technology may be applicable to other antibody-drug conjugate (ADC) therapies.

[0681] Example 6: Stability study of mixed formulations of other ADCs and KN035

[0682] (1)

[0683] Take DS8201 (purchased from Daiichi Sankyo / AstraZeneca, trade name The samples of KN035 (product batch number: 397496) and KN035 were exchanged with a dialysis cartridge, concentrated with an ultrafiltration tube, mixed according to the expected concentration, and prepared into the prescription shown in Table 17.

[0684] Table 17 DS8201 mixed preparation prescription

[0685] After preparation, the samples were placed in a constant temperature and humidity chamber (40±2°C, 70% RH) for 4 weeks, and the insoluble particulate matter content and SEC-HPLC purity of each formulation were tested. The results are shown in Table 18, Figures 16, and 17.

[0686] The test results show that after DS8201 is mixed with different concentrations of KN035, it has good high-temperature stability, no visible foreign matter in the samples, no significant difference in insoluble particles, and the SEC-HPLC aggregate (HMW) content is reduced.

[0687] Table 18 DS8201 mixed preparation test results

[0688] (2)

[0689] Take RC48 (purchased from Rongchang Biotechnology, trade name The samples (product batch numbers: RC48-X1-202206016 / RC48-X1-202311012) and KN035 were exchanged with a dialysis cartridge, concentrated with an ultrafiltration tube, mixed according to the expected concentration, and prepared into the prescription shown in Table 19.

[0690] Table 19 RC48 mixed preparation prescription

[0691] After preparation, the samples were placed in a constant temperature and humidity chamber (40±2°C, 70% RH) for 4 weeks, and the insoluble particulate matter content and SEC-HPLC purity of each formulation were tested. The results are shown in Table 20, Figures 18, and 19.

[0692] The test results showed that after RC48 was mixed with high-concentration KN035, it had good high-temperature stability, no visible foreign matter in the sample, no significant difference in insoluble particles, and reduced SEC-HPLC aggregate (HMW) content.

[0693] Table 20 RC48 mixed preparation test results

Claims

1. A composition, characterized in that Contains programmed death ligand 1 (PD-L1) binding molecules and protein-drug conjugates.

2. The composition according to claim 1, which is a solid preparation, preferably a lyophilized powder.

3. The composition according to claim 1, which is a liquid preparation, preferably an injection.

4. The composition according to any one of claims 1 to 3, wherein the PD-L1 binding molecule comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:

1.

5. The composition of claim 4, wherein the CDR can be a Kabat CDR, an AbM CDR, a Chothia CDR or an IMGT CDR.

6. The composition according to claim 5, wherein 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: 5-7, SEQ ID NO: 8-10, SEQ ID NO: 11-13 and SEQ ID NO: 14-16.

7. The composition according to any one of claims 1 to 6, wherein the PD-L1 binding molecule comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:

1.

8. The composition according to any one of claims 4 to 7, wherein the PD-L1 binding molecule further comprises an immunoglobulin Fc region. 9 . The composition according to claim 8 , wherein the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or a variant thereof. 10 . The composition according to claim 9 , wherein the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO: 2 or SEQ ID NO:

3.

11. The composition according to any one of claims 8 to 10, wherein the immunoglobulin single variable domain is located at the N-terminus of the immunoglobulin Fc region, and the two are directly connected or connected through a peptide chain, and the peptide chain is selected from one or more of the following amino acid sequences: (GS) n 、(GG) n 、(GGS) n 、(GGGS) n 、(GGSG) n 、(GGGGS) n and (GAP) n , wherein n is an integer from 1 to 10.

12. The composition according to any one of claims 1 to 11, wherein the amino acid sequence of the PD-L1 binding molecule is SEQ ID NO:

4.

13. The composition according to any one of claims 1 to 12, wherein the protein-drug conjugate has the following structure: in, P* is antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, Man is mannose, f is 0 or 1, and m is 1 to 10; Gal* is a modified galactose selected from the following structures: LP is: 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 linked to the antibody. The composition according to claim 13 , wherein the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

15. The composition according to claim 13 or 14, wherein the core GlcNAc is linked to the Fc fragment of an antibody; 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.

16. The composition according to any one of claims 13-15, wherein the antibody is a bispecific antibody targeting different epitopes of HER2.

17. The composition of any one of claims 13-16, wherein the antibody has a first heavy chain, a second heavy chain and a common light chain, wherein: The first heavy chain comprises CDR1, CDR2 and CDR3 in the heavy chain variable region of SEQ ID NO: 17, the second heavy chain comprises CDR1, CDR2 and CDR3 in the heavy chain variable region of SEQ ID NO: 18, and the common light chain comprises CDR1, CDR2 and CDR3 in the light chain variable region of SEQ ID NO:

19.

18. The composition according to claim 17, wherein CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO: 17 are selected from any one of the following groups: SEQ ID NO: 23-25, SEQ ID NO: 26-28, SEQ ID NO: 29-31 and SEQ ID NO: 32-34.

19. The composition according to claim 17 or 18, wherein CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO: 18 are selected from any one of the following groups: SEQ ID NO: 35-37, SEQ ID NO: 38-40, SEQ ID NO: 41-43 and SEQ ID NO: 44-46.

20. The composition according to any one of claims 17-19, wherein CDR1, CDR2 and CDR3 in the light chain variable region shown in SEQ ID NO: 19 are selected from any one of the following groups: SEQ ID NO: 47-49, SEQ ID NO: 50-52, SEQ ID NO: 53-55 and SEQ ID NO: 56-58.

21. The composition of any one of claims 16-20, wherein the antibody has a first heavy chain, a second heavy chain, and a common light chain, wherein: The first heavy chain comprises the heavy chain variable region set forth in SEQ ID NO:17, the second heavy chain comprises the heavy chain variable region set forth in SEQ ID NO:18, and the common light chain comprises the light chain variable region set forth in SEQ ID NO:

19.

22. The composition of any one of claims 16-21, wherein the antibody comprises a first heavy chain having an amino acid sequence as shown in SEQ ID NO: 20, a second heavy chain having an amino acid sequence as shown in SEQ ID NO: 21, and a common light chain having an amino acid sequence as shown in SEQ ID NO:

22.

23. The composition according to any one of claims 1 to 22, wherein the concentration of the protein-drug conjugate is 20 mg / mL-150 mg / mL, preferably 50 mg / mL-120 mg / mL, and more preferably 60 mg / mL-100 mg / mL.

24. The composition according to any one of claims 1 to 23, wherein the concentration of the PD-L1 binding protein is 20 mg / mL-180 mg / mL, preferably 50 mg / mL-150 mg / mL, and more preferably 80 mg / mL-120 mg / mL.

25. The composition according to any one of claims 1-24, wherein the ratio of the protein-drug conjugate to the PD-L1 binding molecule is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.

25.

26. The composition according to any one of claims 1 to 25, wherein the pH of the composition is from 4.0 to 7.0, preferably from 4.5 to 6.5, more preferably from 5.0 to 6.

0.

27. The composition according to any one of claims 1 to 26, further comprising a buffer component, wherein the buffer component is selected from one or more of acetic acid-sodium acetate, histidine-histidine hydrochloride and acetate-histidine.

28. The composition according to claim 27, wherein the concentration of the buffer component is 2mM-50mM, preferably 5mM-30mM or 2mM-20mM, more preferably 10mM-20mM or 2mM-10mM.

29. The composition according to any one of claims 1 to 28, further comprising an auxiliary material, wherein the auxiliary material comprises a stabilizer, and the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, proline, glycine, arginine hydrochloride and glycerol, preferably selected from one or more of sucrose, trehalose, mannitol, sorbitol and proline, and more preferably selected from one or more of trehalose, mannitol, sorbitol and proline.

30. The composition according to claim 29, wherein the concentration of the stabilizer is 50 mM-250 mM, preferably 80 mM-200 mM, more preferably 100 mM-150 mM.

31. The composition according to claim 28 or 29, wherein the auxiliary material further comprises an antioxidant, preferably, the antioxidant is methionine.

32. The composition according to claim 31, wherein the concentration of the antioxidant is 10 mM-100 mM, preferably 15 mM-75 mM, more preferably 20 mM-50 mM, even more preferably 30 mM-40 mM.

33. A composition according to any one of claims 28 to 32, further comprising a surfactant selected from the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.

34. The composition of claim 33, wherein the concentration of the surfactant is 0 mg / mL-0.8 mg / mL, preferably 0.05 mg / mL-0.75 mg / mL, more preferably 0.1 mg / mL-0.5 mg / mL, even more preferably 0.2 mg / mL-0.4 mg / mL or 0.1 mg / mL-0.3 mg / mL.

35. A composition according to any one of claims 1 to 34, comprising: (1) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (2) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM sucrose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (3) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM trehalose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (4) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM mannitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (5) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-histidine hydrochloride, 80 mM-200 mM sorbitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (6) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (7) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM sucrose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (8) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM trehalose, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (9) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM mannitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (10) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM acetic acid-sodium acetate, 80 mM-200 mM sorbitol, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (11) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 5 mM-30 mM histidine-acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; (12) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 2 mM-20 mM histidine-acetate, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.0; or (13) 50 mg / mL-120 mg / mL protein-drug conjugate, 50 mg / mL-150 mg / mL PD-L1 binding protein, 2 mM-20 mM histidine-histidine hydrochloride, 80 mM-200 mM proline, 20 mM-50 mM methionine, 0.1 mg / mL-0.5 mg / mL polysorbate 80, pH 5.0 to 6.

0.

36. A method for treating advanced or metastatic solid malignant tumors, characterized in that: Administering the composition of any one of claims 1-35 to a patient in need thereof.

37. A method for treating advanced or metastatic solid malignant tumors, characterized in that: A drug combination of a programmed death-ligand 1 (PD-L1) binding molecule and a protein-drug conjugate is administered to a patient in need thereof, wherein the programmed death-ligand 1 binding molecule is as defined in any one of claims 4-12 and the protein-drug conjugate is as defined in any one of claims 13-22.

38. The method of claim 36 or 37, wherein the malignancy is an advanced / unresectable or metastatic solid malignancy and expresses HER2.

39. The method according to any one of claims 36 to 38, wherein the solid malignant tumor is a gastrointestinal tumor or non-small cell lung cancer.

40. The method according to any one of claims 36-39, wherein the ratio of the protein-drug conjugate to the PD-L1 binding molecule in the composition or drug combination is 1:0.5 to 1:2, preferably 1:0.75 to 1:1.75, more preferably 1:0.8 to 1:1.5, and even more preferably 1:1 to 1:1.

25.

41. The method according to any one of claims 36 to 40, characterized in that: The composition or drug combination is administered to a patient in need thereof once a week, once every two weeks, once every three weeks, or once every four weeks.

42. The method according to any one of claims 36 to 41, characterized in that: The composition or pharmaceutical combination is administered by subcutaneous injection.

43. The method according to any one of claims 36 to 42, wherein the single administration dose of the composition or drug combination is 0.5 mg / kg-10.0 mg / kg, preferably 0.8 mg / kg-7.5 mg / kg, more preferably 1.0 mg / kg-7.0 mg / kg, for example selected from 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg and 6.7 mg / kg.

44. The method according to any one of claims 36 to 43, characterized in that The composition or drug combination is administered to a patient in need thereof by subcutaneous injection once a week, with a single administration dose of 1.1 mg / kg, 2.3 mg / kg, 4.5 mg / kg, 5.6 mg / kg or 6.7 mg / kg.

45. The composition according to any one of claims 1-35, which can be used to treat advanced or metastatic solid malignancies.

46. ​​A drug combination of a programmed death ligand 1 (PD-L1) binding molecule and a protein-drug conjugate, which can be used to treat advanced or metastatic solid malignant tumors; wherein: The programmed death-ligand 1 binding molecule is as defined in any one of claims 4 to 12, and the protein-drug conjugate is as defined in any one of claims 13 to 22.

47. Use of programmed death ligand 1 (PD-L1) binding molecules in combination with protein-drug conjugates in the preparation of drugs for treating advanced or metastatic solid malignant tumors; wherein, The programmed death-ligand 1 binding molecule is as defined in any one of claims 4 to 12, and the protein-drug conjugate is as defined in any one of claims 13 to 22.