Method for preparing antibody-small molecule drug conjugate and use thereof

Abstract

A method for preparing an antigen binding fragment-small molecule drug conjugate and the use thereof. A method for modifying human IgG Fc comprises modifying the N-terminal sequence of human IgG Fc. When fusing the obtained IgG Fc variant and an antigen binding fragment and expressing same, the loading capacity of the functional molecule is remarkably increased, and the polymer generation following expression is greatly reduced. Also provided are a fusion protein comprising the IgG Fc variant, and a drug conjugate.

Description

Method for preparing antibody-coupled small molecule drugs and application thereof

[0001] This application claims priority to patent application filed on July 27, 2023, application number CN202310929139.3; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of biotechnology and pharmacology, and more specifically, relates to a method for preparing an antibody-coupled small molecule drug and its application. Background Art

[0003] Antibody-drug conjugates (ADCs) are chemically linked to small-molecule drugs via antibodies. Leveraging the targeting properties of antibodies, they deliver the small-molecule drug to target cells, thereby exerting its therapeutic effect. ADCs, by combining the dual advantages of the targeting properties of antibodies with the high activity of small-molecule drugs in tumor tissue, are highly effective in killing tumor cells, with fewer side effects than traditional chemotherapy drugs and greater efficacy than traditional antibody-based cancer drugs. They have been dubbed "biological missiles" in the field of cancer treatment, demonstrating immense clinical therapeutic value and becoming a research hotspot in cancer treatment.

[0004] At present, the coupling process of most antibody-drug conjugates relies on the random or fixed-point coupling of small molecule drugs with amino acid residues in antibodies, among which random coupling is the main method. Random coupling methods include, for example, lysine residue coupling and cysteine ​​residue coupling. Lysine residue coupling is the use of lysine residues in antibodies for coupling. There are more than 80 lysine residues in IgG molecules, of which more than 20 sites are highly solvent accessible. These lysine residues can be coupled to small molecule drugs through acylation. However, due to the large number of sites that can be coupled, it is easy to cause the product heterogeneity of each batch of antibody-drug conjugates to be large, and the drug-antibody ratio (DAR) is unevenly distributed, which in turn affects the drug dynamics and drug metabolism of antibody-drug conjugates. Cysteine ​​residue coupling is the use of cysteine ​​residues in antibodies for coupling. There are generally no free cysteine ​​residues on the surface of antibodies that can be used for coupling reactions. They all exist in the form of disulfide bonds.

[0005] Human IgG can be divided into four subtypes: IgG1, IgG2, IgG3, and IgG4. The Fc fragments of different IgG subtypes differ in structure and function due to differences in the location of interchain disulfide bonds and molecular weight. The vast majority of ADCs currently on the market and under development are conventional IgGs, and most utilize the human IgG1 subtype.

[0006] Heavy-chain antibodies, originally discovered in camels and sharks, are a new type of antibody molecule composed solely of heavy chains, devoid of light chains. While lacking light chains, heavy-chain antibodies retain the ability to bind antigens. They also offer advantages such as small molecular weight, ease of expression, engineering, and excellent stability. Consequently, they are increasingly being used in the development of antibody-based drugs. However, there are currently no reports of successful commercialization of antibody-drug conjugates using heavy-chain antibodies.

[0007] Antibody-drug conjugates (ADCs) are prepared using heavy-chain antibodies to conjugate small molecule drugs to the antibody via cysteine ​​residues. However, if the Fc of human IgG1 is used, the interchain disulfide bonds that can be utilized are very limited. In the art, a single ADC molecule can only be conjugated to a maximum of four small molecule drugs. However, to improve drug efficacy, more small molecule drugs must be conjugated, for example, 6-8 small molecule drugs.

[0008] Therefore, this field urgently needs further optimization and modification of such antibody-drug conjugates in order to increase the amount of small molecule drugs carried and improve drug efficacy.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a method for preparing an antibody-coupled small molecule drug and its application.

[0011] In a first aspect of the present invention, a method for increasing the functional molecular load of an antigen binding fragment-human IgG Fc fusion protein and reducing the production of polymers after expression is provided, comprising: (1) providing an antigen binding fragment-human IgG Fc fusion protein, wherein the N-terminal sequence of the human IgG Fc is a modified sequence, wherein the modification is: replacing 6-15 (preferably 7-13 or 8-12) amino acid residues at the N-terminus with the amino acid residues shown in SEQ ID NO: 6 (ERKCCVECPPCP), and based on the sequence, performing a modification selected from the following group: (a) inserting 1-9 amino acid residues between, before (N-terminus) or after (C-terminus) the CC residues at positions 4 and 5 of the sequence of SEQ ID NO: 6; (b) mutating either C at position 4 or 5 of the sequence of SEQ ID NO: 6.

[0012] In one or more embodiments, in (a), 2-8 non-cysteine ​​residues (preferably 2-7 residues; more preferably 2-4 residues) are inserted between the two CC residues.

[0013] In one or more embodiments, the amino acid residues for insertion or mutation modification include aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine or histidine.

[0014] In one or more embodiments, the amino acid residue used for insertion is an acidic amino acid, including aspartic acid, glutamic acid, or a combination thereof.

[0015] In one or more embodiments, the method further comprises: (2) linking functional molecules to the antigen binding fragment-human IgG Fc fusion protein; the loading amount of the functional molecules is 6-8.

[0016] In one or more embodiments, the functional molecules include: small molecule anti-tumor drugs, cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

[0017] In one or more embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.

[0018] In one or more embodiments, the tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin.

[0019] In one or more embodiments, the detectable markers include: fluorescent markers and colorimetric markers.

[0020] In one or more embodiments, the anti-tumor toxins include: toxins that act on tubulin; toxins that act on DNA; compounds that act on intracellular metabolism, transcription, translation or signal transduction.

[0021] In one or more embodiments, the anti-tumor cytokine comprises IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof.

[0022] In one or more embodiments, the antibody that binds to a tumor surface marker is an antibody that recognizes a tumor antigen.

[0023] In one or more embodiments, the toxin acting on tubulin is monomethyl auristatin, its related compounds or derivatives thereof.

[0024] In one or more embodiments, the toxin acting on tubulin is a maytansinoid, a related compound thereof, or a derivative thereof.

[0025] In one or more embodiments, the DNA-acting toxin is duocarmycin, calicheamicin, pyrrolobenzodiazepines, SN-38, DXd, related compounds or derivatives thereof.

[0026] In one or more embodiments, the anti-tumor toxin is connected to the fusion protein via a linker, and the linker includes: a non-cleavable linker; a cleavable linker, including a disulfide bond-based linker, a tissue protease-sensitive linker, a hydrazone bond-based linker, and a lysosomal protease-based linker.

[0027] In one or more embodiments, the non-cleavable linker is SMCC.

[0028] In one or more embodiments, the disulfide bond-based linker is sulfo-SPDB.

[0029] In one or more embodiments, the cathepsin-sensitive linker is mc-vc-PAB.

[0030] In one or more embodiments, the lysosomal protease-based linker is a maleimide-GGFG peptide linker.

[0031] In one or more embodiments, the intracellular signaling region includes: CD3ζ chain, FcεRIγ tyrosine activation motif, and intracellular signaling regions of costimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, and CD40.

[0032] In one or more embodiments, the transmembrane region includes the transmembrane region of CD8 or CD28.

[0033] In one or more embodiments, the human IgG is selected from: human IgG1, human IgG2 or human IgG4.

[0034] In one or more embodiments, the antigen-binding fragment includes (but is not limited to): a polypeptide capable of binding to an antigen in a heavy chain antibody, a single chain antibody, a single domain antibody, a BiTE antibody, a DART antibody, an Fv antibody, and an Fd antibody.

[0035] In one or more embodiments, the antigen-binding fragment is the variable region of the heavy chain or light chain of an antibody, or a combination of the two.

[0036] In another aspect of the present invention, a modified human IgG Fc variant is provided, wherein the N-terminal sequence of the human IgG Fc is a modified sequence, wherein the N-terminal 6-15 (preferably 7-13 or 8-12) amino acid residues are replaced with the amino acid residues shown in SEQ ID NO: 6 (ERKCCVECPPCP), and based on this sequence, modifications are made selected from the following groups: (a) inserting 1-9 amino acid residues between, before (N-terminus) or after (C-terminus) the CC residues at positions 4 and 5 of the sequence of SEQ ID NO: 6; (b) mutating either C at position 4 or 5 of the sequence of SEQ ID NO: 6.

[0037] In one or more embodiments, in the engineered human IgG Fc variant, 2-8 amino acid residues (preferably 2-7 amino acid residues; more preferably 2-4 amino acid residues) are inserted between the two CC residues.

[0038] In one or more embodiments, the amino acid residues for insertion or mutation modification in the engineered human IgG Fc variant include aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine or histidine.

[0039] In one or more embodiments, in the engineered human IgG Fc variant, the amino acid residue used for insertion is an acidic amino acid, including aspartic acid, glutamic acid, or a combination thereof.

[0040] In one or more embodiments, in the engineered human IgG Fc variant, the N-terminal sequence of the human IgG Fc is an engineered sequence, and the engineering comprises mutating the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, or SEQ ID NO: 7.

[0041] In another aspect of the present invention, there is provided a use of any of the aforementioned modified human IgG Fc variants for preparing an antigen-binding fragment-human IgG Fc fusion protein; when functional molecules are linked to the antigen-binding fragment-human IgG Fc fusion protein, the functional molecule loading capacity is 6-8.

[0042] In another aspect of the present invention, a fusion protein is provided, which is an antigen-binding fragment-human IgG Fc fusion protein; wherein the human IgG Fc is any one of the human IgG Fc variants described above.

[0043] In another aspect of the present invention, a polynucleotide is provided, which encodes any of the above-mentioned human IgG Fc variants, or encodes any of the above-mentioned fusion proteins.

[0044] In another aspect of the present invention, an expression construct (such as an expression vector) is provided, which comprises the polynucleotide.

[0045] In another aspect of the present invention, an expression system is provided, which comprises the expression construct or the polynucleotide integrated into the genome.

[0046] In another aspect of the present invention, a method for preparing any of the above-mentioned human IgG Fc variants or fusion proteins is provided, comprising: expressing using the expression system under conditions suitable for expression, thereby obtaining the human IgG Fc variant or fusion protein.

[0047] In another aspect of the present invention, a conjugate is provided, comprising: the fusion protein described above; and a functional molecule connected to the fusion protein; the loading amount of the functional molecule is 6-8.

[0048] In one or more embodiments, the functional molecules include: small molecule anti-tumor drugs, cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

[0049] In another aspect of the present invention, a pharmaceutical composition or a drug kit is provided, comprising: the fusion protein or the conjugate.

[0050] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is an SDS-PAGE image of a heavy chain antibody constructed using human IgG1 Fc.

[0052] FIG2 is an SDS-PAGE image of the heavy chain antibody after IgG1 Fc mutation.

[0053] FIG3 is an SDS-PAGE image of the heavy chain antibody after IgG1 Fc cysteine ​​mutation.

[0054] FIG4 is an SDS-PAGE image of heavy chain antibodies in which different numbers of amino acids were inserted between the cysteine ​​residues at positions 4 and 5 of IgG1 Fc.

[0055] FIG5 is an SDS-PAGE image of a heavy chain antibody in which amino acids were inserted before, in, and after the cysteine ​​residue of IgG1 Fc.

[0056] FIG6 is an SDS-PAGE image of a heavy chain antibody in which amino acids of different properties are inserted between the Fc region of IgG1.

[0057] Figure 7 shows the SEC results of the heavy chain antibody conjugated with the small molecule DXd by inserting amino acids with different properties into IgG1 Fc.

[0058] FIG8 is an SDS-PAGE image of heavy chain antibodies after mutations at the corresponding positions of Fc of human IgG2 and IgG4.

[0059] FIG9 shows the SEC results of coupling the heavy chain antibodies with mutations at the corresponding positions of the Fc of human IgG2 and IgG4 to the small molecule DXd.

[0060] Figure 10 shows the binding ability of IgG1 Fc mutated heavy chain antibodies and heavy chain antibody-drug conjugates to OVCAR-3 cells.

[0061] FIG11 shows the killing effect of antibody-drug conjugates prepared from IgG1 Fc mutated heavy chain antibodies on OVCAR-3 cells. DETAILED DESCRIPTION

[0062] After extensive research and experimentation, the present inventors have discovered a method for modifying human IgG Fc, including modifying the N-terminal sequence of human IgG Fc. When the IgG Fc variant obtained by the present invention is fused with an antigen-binding fragment and expressed, the loading capacity of the functional molecule is significantly increased and the formation of aggregates after expression is greatly reduced. The present invention also discloses fusion proteins and drug conjugates (such as antibody-drug conjugates) comprising the IgG Fc variant.

[0063] As used herein, the "antigen-binding fragment" or "antigen-binding site" or "antigen-binding portion" of an antibody refers to the key region in the antibody that determines the binding of the antibody to the antigen, which can be the shortest sequence segment that determines the binding of the antibody to the antigen.

[0064] As used herein, the terms "heavy chain antibody", "single domain antibody", "domain antibody" or "nanobody" are used interchangeably.

[0065] As used herein, "operably linked" or "operably linked to" refers to a situation in which certain portions of a linear DNA sequence / amino acid sequence are organically linked and cooperate with other portions of the same linear DNA sequence / amino acid sequence. For example, if a promoter controls the transcription of a coding sequence, it is operably linked to a coding sequence.

[0066] As used herein, the terms “contain,” “have,” or “include” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0067] As used herein, a "pharmaceutically acceptable carrier," "physiologically acceptable carrier," or "biologically acceptable carrier" is a solvent, suspending agent, or excipient, etc., used to deliver the fusion protein of the present invention to an object (including food or feed) requiring treatment, and is environmentally friendly or harmless to humans and animals, with controllable toxicity and side effects. The carrier can be liquid or solid, and preferably is one that can maintain the biological activity of the fusion protein of the present invention to a high degree.

[0068] IgG Fc modification

[0069] The inventors' previous research revealed that when constructing fusion proteins using the Fc fragment of human IgG and the antigen-binding fragment (AF), a large number of aggregates are easily formed during expression, affecting the quality of the fusion protein and limiting the amount of functional molecules that can be loaded into the fusion protein. Through in-depth research and analysis, the inventors modified the IgG Fc, increasing the functional molecule loading capacity of the AF-human IgG Fc fusion protein and preventing aggregate formation. This, in turn, offers promising prospects for the development of fusion proteins with IgG and Fc.

[0070] Therefore, the present invention provides a novel method for modifying IgG Fc, comprising modifying the N-terminal sequence of human IgG Fc by replacing a portion of the amino acid residues at the N-terminus with the amino acid residues set forth in SEQ ID NO: 6, and performing modifications based on this sequence selected from the group consisting of: (a) inserting 1-9 amino acid residues between, before (N-terminus), or after (C-terminus) the C-C residues at positions 4 and 5 of SEQ ID NO: 6; and (b) mutating either C at position 4 or 5 of SEQ ID NO: 6. The modifications yield IgG Fc variants capable of well-linking to multiple functional molecules; the resulting antigen-binding fragment-human IgG Fc fusion proteins exhibit significantly reduced or eliminated aggregate formation during expression.

[0071] The IgG Fc variants (mutants) of the present invention are synthetic or recombinant proteins, i.e., they can be chemically synthesized or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants). Depending on the host used in the recombinant production protocol, the IgG Fc variants of the present invention may or may not include an initial methionine residue.

[0072] The present invention also includes fragments (biologically active fragments), derivatives and analogs of IgG Fc variants. As used herein, the terms "fragment," "derivative," and "analog" refer to proteins that substantially retain the same biological function or activity as the mutant proteins listed in the Examples of the present invention.

[0073] The fragments, derivatives or analogs of the IgG Fc variants of the present invention can be, based on the IgG Fc variants specifically listed in the present invention: (i) a mutein having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutein having a substitution group in one or more amino acid residues, or (iii) a mutein formed by fusion of a mature mutein with another compound (such as a compound that extends the half-life of the mutein, such as polyethylene glycol), or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence (such as a leader sequence or secretory sequence or a sequence used to purify the mutein or a proprotein sequence, or a fusion protein formed with an antigenic IgG fragment). However, in the amino acid sequences of the fragments, derivatives and analogs, corresponding to wild-type IgG Fc, the key mutation sites / regions claimed in the present invention (such as the sequence regions shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, and SEQ ID NO: 7 at the N-terminus) are conserved; that is, the sequence changes covered by the fragments, derivatives and analogs are present in non-critical positions on IgG Fc, and the fragments, derivatives and analogs can still have the same or substantially the same functions / activities as the IgG Fc variants listed in the examples of the present invention.

[0074] Furthermore, the IgG Fc variants of the present invention may also be modified. Modifications (generally without altering the primary structure) include in vivo or in vitro chemical derivatization such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during the synthesis and processing of the mutant protein or during further processing steps. Such modifications can be achieved by exposing the protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are derivative proteins modified to enhance resistance to proteolysis, increase half-life, or optimize solubility. Similarly, in the amino acid sequence of the modified IgG Fc variant, the key mutation sites / regions claimed in the present invention are conserved relative to wild-type IgG Fc; that is, the sequence changes encompassed by the fragments, derivatives, and analogs are present at non-critical locations on the IgG Fc, and the fragments, derivatives, and analogs can still exhibit the same or substantially the same functions / activities as the IgG Fc variants listed in the Examples of the present invention.

[0075] The term "polynucleotide encoding a mutant protein" may include a polynucleotide encoding the IgG Fc variant of the present invention, or may also include additional coding and / or non-coding sequences.

[0076] Fusion protein

[0077] The IgG Fc variant of the present invention can be fused with the antigen-binding fragment of an antibody to form an antigen-binding fragment-human IgG Fc fusion protein.

[0078] In the novel fusion protein of the present invention, the antigen binding fragment and human IgG Fc are operably connected, and the two have ideal compatibility and high biological activity.

[0079] As a preferred embodiment of the present invention, the IgG Fc and the antigen-binding fragment are connected via a polypeptide linker to form a fusion protein. For example, the linker comprises 3-40 amino acids. As a commonly used linker, the amino acid sequence of the linker is (GSG) n or (GGGGS) n or (GGGS) n (For example, n=1 to 8).

[0080] The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (ie, "antigen-binding portion") or single chain thereof.

[0081] "Monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0082] "Antigen-binding fragment" refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen (e.g., PD-1). It is understood that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of the VH domain; and (vi) isolated complementarity determining regions (CDRs).

[0083] The antibodies of the present invention include humanized antibodies, and "humanized antibodies" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0084] The antibodies of the present invention include chimeric antibodies. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0085] As a more preferred embodiment of the present invention, in the fusion protein, the IgG Fc is linked to the variable region of a heavy chain antibody (VHH), thereby forming the variable region of the heavy chain antibody - human IgG Fc. A "heavy chain antibody" is an antibody comprising a heavy chain, the antigen-binding portion of which contains only the heavy chain variable region and does not contain a light chain and its variable region.

[0086] Nucleic acid molecules and constructs or cells containing the same

[0087] The present invention also provides an isolated nucleic acid encoding the human IgG Fc variant or the fusion protein, or a complementary chain thereof.

[0088] Furthermore, the present invention provides a vector comprising a nucleic acid molecule encoding the human IgG Fc variant or fusion protein. The vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the human IgG Fc variant or fusion protein.

[0089] A variety of suitable nucleic acid molecules encoding human IgG Fc variants are suitable for use in the present invention. A variety of suitable nucleic acid molecules encoding human IgG Fc variants or fusion proteins are also suitable for use in the present invention. The sequences mentioned in the examples below are all suitable for use in the methods of the present invention. It should be understood that once the amino acid sequence of a protein is provided, one skilled in the art can readily determine the nucleic acid molecule encoding it.

[0090] A variety of suitable vectors can be used, such as those used for cloning and expression in mammals, bacteria, fungi, and yeast, as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier latest edition). In a preferred embodiment of the present invention, the vector is a mammalian cell-compatible vector.

[0091] In some embodiments, the vector may be some viral vectors, such as but not limited to retroviral vectors, phage vectors, adenoviral vectors, herpes simplex virus (HSV) vectors, AAV vectors, or lentiviral vectors.

[0092] Expression vectors include human IgG Fc variants or fusion protein DNA sequences that are connected with suitable transcription and translation regulatory sequences, such as mammals, microorganisms, viruses or insect genes. Regulatory sequences include transcription promoters, operators, enhancers, ribosome bind sites or suitable sequences that control transcription and translation initiation and termination. When IgG Fc variants or fusion protein sequences need regulatory sequence functions, suitable regulatory sequences are connected. Like this, the promoter sequence is connected to the DNA sequence front end of encoding human IgG Fc variants or fusion protein. The ability to replicate in the host cell is usually controlled by the replication origin. The screening gene that is used for transformant identification also can be added to the expression vector.

[0093] Alternatively, the leader sequence can be fused to the coding sequence for the human IgG Fc variant or fusion protein, thereby enabling extracellular secretion of the translated human IgG Fc variant or fusion protein. The signal peptide can enhance extracellular secretion of the chimeric polypeptide by the host cell. The signal peptide can be cleaved during secretion of the polypeptide from the cell.

[0094] The present invention also provides an expression system (e.g., a host cell) for expressing a human IgG Fc variant or fusion protein, the expression system comprising the expression vector of the present invention or an exogenous polynucleotide of the present invention integrated into the genome. Any cell suitable for expression by an expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to Escherichia coli; Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast, filamentous fungi, plant cells; Drosophila S2 or SF9 insect cells; CHO, COS, HEK293 cells, or BOWES melanoma cells, etc. Methods for constructing the expression system should be known to those skilled in the art, for example, can include but not limited to a combination of one or more of microinjection, gene gun method, electroporation method, virus-mediated transformation method, electron bombardment method, calcium phosphate precipitation method, etc. As a preferred embodiment of the present invention, the expression system is an animal cell expression system, such as CHO, NS0, BHK, HEK-293 or PER-C6, etc. In a more specific embodiment, the cell is CHO (such as CHO-K1, CHO-S, CHO-DG44, ExpiCHO, CHOZN, etc.).

[0095] Methods for producing human IgG Fc variants or fusion proteins are also encompassed by the present invention. The methods comprise culturing recombinant cells containing nucleic acid encoding the human IgG Fc variant or fusion protein. The methods may include allowing the cells to express the encoded human IgG Fc variant or fusion protein, and renaturing the expressed human IgG Fc variant or fusion protein. In one embodiment, the methods may further comprise isolating and / or purifying the renatured human IgG Fc variant or fusion protein. The products of the methods are also protected.

[0096] The human IgG Fc variant or fusion protein prepared above can be purified to substantially homogeneous properties, for example, to appear as a single band on SDS-PAGE electrophoresis.

[0097] Conjugated drugs

[0098] The technical solution of the present invention is a molecular design and improvement of biomacromolecules, which can be applied to the development of biomacromolecule drugs.

[0099] The fusion proteins of the present invention can be further linked or coupled with other heterologous functional molecules to form conjugates (e.g., antibody-drug conjugates (ADCs)); the heterologous functional molecules may include, but are not limited to, cytokines, detectable markers, toxins (e.g., tumor suppressor toxins), transcriptional activation domains, transcriptional repression domains, nucleases, deaminases, methylases, demethylases, transcriptional release factors, and the like. The detectable markers may include, but are not limited to, fluorescent markers, chromogenic markers, reporter genes, localization signals, and the like. The heterologous functional domains may be linked, coupled, or conjugated to the N-terminus, C-terminus, or internally of the fusion proteins of the present invention.

[0100] The detectable label may include, but is not limited to, fluorescent labels and chromogenic labels, such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More than one label may be included. The label used to label the antibody for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnostic technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection / analysis / diagnostic techniques and / or methods known in the art are well known to those skilled in the art.

[0101] The technical solutions of the present invention can be applied to the preparation of antibody-drug conjugates (ADCs). Fusion proteins of the present invention carrying the VHH variable region can be combined with small molecule drugs via linkers to produce ADCs. These ADCs combine the powerful lethality of small molecule drugs with the targeting properties of monoclonal antibodies. Leveraging the high specificity of antibodies, they precisely deliver the drug to tumor cells, avoiding damage to normal cells in the body and thus reducing adverse reactions during treatment. ADCs have become a promising class of anticancer drugs and are one of the fastest-growing areas of cancer treatment.

[0102] Compared to traditional antibodies, heavy-chain antibodies (HcAbs) consist of only two heavy chains and no light chains, and have the characteristics of small molecular weight, high expression level, and good stability. Heavy-chain antibodies can also be regarded as an Fc fusion protein, whose heavy chain is composed of the VHH variable region that binds to the antigen and the conventional Fc structural region. In the field, how to modify human IgG Fc, increase its amount of targeted drug assembly, reduce the formation of aggregates, and how to successfully apply heavy-chain antibodies with human IgG Fc in drug development are key technologies that need to be solved urgently. The present invention provides a new solution. The modified human IgG Fc can be used to construct heavy-chain antibodies.

[0103] In the present invention, the human IgG may include human IgG1, human IgG2, or human IgG4. Heavy chain antibodies with a human IgG2 Fc have significantly weaker ADCC and CDC capabilities than those with an IgG1 Fc. The use of a human IgG2 Fc provides an alternative for heavy chain antibodies that do not require ADCC and CDC. Heavy chain antibodies with a human IgG2 Fc are also prone to aggregates for the same reasons as fusion proteins, making the modifications described in the present invention particularly important, facilitating process development for heavy chain antibody drugs with a human IgG2 Fc.

[0104] As demonstrated in the examples of the present invention, using the optimized and modified human IgG Fc of the present invention, a single ADC molecule can be coupled to six or eight small-molecule drugs, significantly improving the ADC's ability to carry small-molecule drugs and thereby enhancing its ability to kill target cells (e.g., tumor cells). However, the process of preparing ADCs using human IgG Fc heavy chain antibodies also faces the problem of aggregate formation. Therefore, the preferred mutations of the present invention reduce aggregate formation, making process development easier for antibodies with human IgG Fc heavy chains. After preparation into ADCs, their functionality is superior to that of IgG1 heavy chain antibodies.

[0105] Pharmaceutical composition or kit

[0106] The present invention also provides a pharmaceutical composition comprising: a fusion protein / conjugate / adjuvant (e.g., an antibody-drug conjugate) of the present invention, or a polynucleotide encoding the same, or an expression vector containing the polynucleotide, or an expression system (e.g., a recombinant cell) expressing the fusion protein / conjugate / adjuvant; and a pharmaceutically or physiologically acceptable carrier. Unless otherwise indicated, " / " represents "or."

[0107] Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A comprehensive description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in compositions may include liquids such as water, phosphate buffered saline, Ringer's solution, physiological saline, balanced salt solutions, lyoprotectants such as glycerol or sorbitol, and the like. In addition, these carriers may also contain auxiliary substances such as lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and stabilizers such as albumin.

[0108] During use, a safe and effective amount of the fusion protein / conjugate / adjuvant (e.g., antibody-drug conjugate) of the present invention, or a polynucleotide encoding the same, or an expression vector containing the polynucleotide, or an expression system expressing the fusion protein / conjugate / adjuvant, is administered to a mammal (e.g., a human). The safe and effective amount is generally at least about 0.001 μg / kg body weight, and in most cases does not exceed about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0109] The precise effective amount for a subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. For a given condition, the effective amount can be determined using routine experiments, which is within the judgment of the clinician.

[0110] The present invention also provides a drug box or a test kit, comprising: the fusion protein / conjugate / coupled substance (such as an antibody-drug conjugate) of the present invention or the polynucleotide encoding them, or an expression vector containing the polynucleotide or an expression system expressing the fusion protein / conjugate / coupled substance; or the pharmaceutical composition.

[0111] For ease of clinical application, the pharmaceutical composition of the present invention can be contained in an injection device (such as an injection needle), which can contain a single dose of the pharmaceutical composition. The injection device can be contained in a medicine box for easy storage and use.

[0112] The medicine box or test kit of the present invention may also include instructions for use to facilitate those skilled in the art to use it in the correct manner.

[0113] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Science Press, or according to the conditions recommended by the manufacturer.

[0114] Example 1. Preparation of heavy chain antibody-drug conjugates

[0115] To obtain heavy chain antibody-drug conjugates, the variable region encoding genes of two different heavy chain antibodies (VHH1 and VHH2) were selected and ligated with the gene encoding the Fc of human IgG1. The ligated genes were then constructed into the multiple cloning site of the pCGS3 (Merck) expression vector. The constructed plasmid was then transfected into ExpiCHO cells for expression. The heavy chain antibodies were purified by Protein A and were designated Antibody 1 and Antibody 2, respectively. The amino acid sequence of the human IgG1 Fc is as follows (SEQ ID NO: 1):

[0116] The amino acid sequences of the two different heavy chain antibody variable regions are as follows:

[0117] VHH1 (SEQ ID NO: 2):

[0118] VHH2 (SEQ ID NO: 3):

[0119] Protein purity was assessed by SDS-PAGE. Under non-reducing conditions, the molecular weight was approximately 80 kDa. As shown in Figure 1, a band with a molecular weight of approximately 80 kDa appeared for Antibody 1 and Antibody 2 under non-reducing conditions, indicating that no aggregates were formed after the heavy chain variable region of the antibody was linked to the Fc of human IgG1, and that the antibody purity was high.

[0120] Antibody 1 and Antibody 2 were incubated with TCEP (Sigma) to break the disulfide bond between the two heavy chains. Maleimide-GGFG-DXd (MedChemExpress) was then added for conjugation. Finally, the conjugated products were purified using a desalting column to remove excess small molecule drug. Hydrophobic interaction chromatography and LC-MS analysis revealed a 1:4 ratio of conjugated drug to small molecule for both Antibody 1 and Antibody 2.

[0121] Example 2: Effect of mutation on polymer formation

[0122] Since the heavy chain antibody in Example 1 can be coupled to a maximum of four small molecules when coupled to small molecule drugs, it would be advantageous if it could be modified to load more small molecules. The inventors conducted in-depth sequence analysis and experimental research, and the results showed that mutating a portion of the IgG1 sequence (positions 1-10) can achieve the goal of increasing disulfide bonds. The corresponding mutation sites and mutation schemes are shown in Table 1.

[0123] Table 1

[0124] The mutated sequence is ERKCCVECPPCP (SEQ ID NO: 6). That is, the variable region genes of two different heavy chain antibodies, VHH1 and VHH2, are connected to the mutated IgG1 Fc coding gene.

[0125] Specifically, the gene encoding the corresponding position in the human IgG1 Fc sequence was first replaced with the mutated gene. The VHH1 or VHH2 encoding genes for two different heavy-chain antibody variable regions were then ligated to the 5' end of the IgG1 Fc gene. The ligated genes were then constructed into the multiple cloning site of the pCGS3 (Merck) expression vector. The constructed plasmid was then transfected into ExpiCHO cells for expression. The heavy-chain antibodies were purified by Protein A. The antibodies with VHH1 or VHH2 ligated to the mutated Fc were designated Antibody 3 and Antibody 4, respectively. Protein purity was assessed by SDS-PAGE.

[0126] As shown in Figure 2, in the non-reduced state, the two antibodies, in addition to having a band corresponding to the target molecular weight, also have bands with larger molecular weights, especially a very wide band corresponding to twice the target molecular weight. This shows that after the Fc of human IgG1 was mutated accordingly, a large number of aggregates appeared in the expressed heavy chain antibodies, especially dimers. Moreover, aggregates appeared in two different heavy chain antibodies, indicating that this is a common phenomenon. The results suggest that this may be due to the presence of interchain disulfide bonds in the mutation target region, which is prone to mispairing during the expression of heavy chain antibodies, resulting in the generation of aggregates. Therefore, the inventors decided to make further modifications.

[0127] The present inventors mutated the cysteine ​​residues (C) at positions 4, 5, 8, and 11 of the mutated sequences in Table 1 to other amino acid residues to reduce the formation of a pair of disulfide bonds. The present inventors mutated the cysteine ​​residues (C) at positions 4, 5, 8, and 11 to amino acid residues with similar side chain sizes to the cysteine ​​residues, exemplified by the mutation to serine residues (S), as shown in Table 2.

[0128] The sequence of the corresponding position of IgG1 of antibody 3 (corresponding to the position shown in Table 1) was mutated as shown in Table 2, and the constructed plasmid was transfected into ExpiCHO cells for expression. Mutant 1, mutant 2, mutant 3 and mutant 4 were obtained by Protein A purification.

[0129] Under non-reduced conditions, the molecular weight of the mutated heavy chain antibody is approximately 80 kDa. As shown in FIG3 , under non-reduced conditions, the aggregates of mutants 1, 2, 3, and 4 are significantly reduced, especially after mutations at positions 4 and 5, where there are almost no aggregates.

[0130] The results of size exclusion chromatography analysis are shown in Table 2, which further illustrate that changes in the positions of the cysteine ​​residues at positions 4, 5, 8, and 10 can significantly reduce the formation of aggregates, especially changes in the cysteine ​​residues at positions 4 and 5 significantly reduce the formation of aggregates.

[0131] Table 2

[0132] Analysis of expression yields showed that the expression levels of Antibody 1, Antibody 2, and Mutants 1-4 were all in the range of 120-150 μg / mL, indicating that the mutations did not affect the expression level of the heavy chain antibody.

[0133] Example 3: Effect of inserting different numbers of amino acids into the heavy chain antibody mutation region on polymer formation

[0134] As can be seen from Mutant 1, Mutant 2, Mutant 3 and Mutant 4 in Example 2, the heavy chain antibody after sequence mutation not only significantly reduces the production of aggregates, but also increases the number of interchain disulfide bonds of the heavy chain antibody from 2 pairs to 3 pairs, thereby increasing the number of coupled small molecules from 4 to 6.

[0135] To further increase interchain disulfide bonds, the inventors conducted in-depth research and experiments and found that another modification method was effective: inserting different numbers of amino acid residues between the cysteine ​​residues at positions 4 and 5 of the aforementioned mutation positions. To verify the effect of inserting different numbers of amino acid residues, the inventors inserted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 amino acid residues, respectively. Here, the insertion of alanine residue (A) is used as an example, as shown in Table 3.

[0136] The above 13 different mutants were connected to the C-terminus of the VHH1 encoding gene, and mutants 5 to 17 were obtained by expression and purification.

[0137] The SDS-PAGE results are shown in Figure 4 and Table 3. Inserting different numbers of alanine residues (A) between the cysteine ​​residues at positions 4 and 5 can reduce the formation of aggregates. Inserting 2, 4, 5, 6, or 7 alanine residues (A) completely eliminates the formation of aggregates. This indicates that inserting 2, 4, 5, 6, or 7 amino acid residues between the cysteine ​​residues at positions 4 and 5 can greatly reduce the formation of heavy chain antibody aggregates.

[0138] Table 3

[0139] In order to study whether inserting amino acid residues before and after the cysteine ​​residues at positions 4 and 5 can also reduce the formation of aggregates, two amino acid residues were inserted before or after the cysteine ​​residues at positions 4 and 5, respectively. Here, the insertion of two alanine residues (A) is taken as an example, as shown in Table 4.

[0140] The IgG Fc encoding genes with different mutation forms listed in Table 4 were ligated to the 3' end of the VHH1 encoding gene, respectively. The ligated genes were constructed into the pCGS3 (Merck) expression vector. The constructed plasmids were then transfected into ExpiCHO cells for expression, and mutants 18 and 19 were obtained by Protein A purification.

[0141] The results are shown in Figure 5. Compared with antibody 3, mutants 6, 18 and 19 can reduce the formation of aggregates by inserting alanine residues (A) before, in the middle and after the cysteine ​​residues at positions 4 and 5. The results of size exclusion chromatography analysis are shown in Table 4. Inserting two amino acids between the cysteine ​​residues at positions 4 and 5 has the most obvious effect on reducing the formation of aggregates.

[0142] Table 4

[0143] The above results indicate that inserting amino acids before, in, or after the cysteine ​​residues at positions 4 and 5 can significantly reduce the formation of heavy chain antibody aggregates, but inserting amino acid residues between the cysteine ​​residues at positions 4 and 5 has the best effect.

[0144] Example 4: Effects of Inserting Amino Acids of Different Properties into the Mutation Target Region on Antibody-Drug Conjugates

[0145] Based on Antibody 3 and Antibody 4, amino acid residues of different properties were inserted between the cysteine ​​residues at positions 4 and 5, including non-polar amino acids (here, exemplified by the insertion of two alanine residues (A); polar uncharged amino acids (here, exemplified by the insertion of two serine residues (S); basic amino acids (here, exemplified by the insertion of two lysine residues (K); and acidic amino acids (here, exemplified by the insertion of two glutamic acid residues (E) and two aspartic acid residues (D), as shown in Table 5). Different mutants were expressed and purified. As shown in Figure 6, the insertion of amino acid residues of different properties between the cysteine ​​residues at positions 4 and 5 significantly reduced aggregate formation compared to Antibody 3 and Antibody 4. Size exclusion chromatography analysis, as shown in Table 5, showed that Mutant 6 and Mutants 20-28 produced almost no aggregates.

[0146] Table 5

[0147] The above-mentioned heavy chain antibodies with different mutations were incubated with TCEP (Sigma) to open the disulfide bonds between the chains, and then maleimide-GGFG-DXd (MedChemExpress) was added for coupling. Finally, they were purified using a desalting column to remove excess small molecule drugs. The obtained antibody-drug conjugates were named Antibody 3-DXd, Mutant 6-DXd, Mutant 20-DXd, Mutant 21-DXd, Mutant 22-DXd, Mutant 23-DXd, Antibody 4-DXd, Mutant 24-DXd, Mutant 25-DXd, Mutant 26-DXd, Mutant 27-DXd and Mutant 28-DXd.

[0148] The results showed that antibodies 3-DXd and 4-DXd produced significant precipitation during preparation. The inventors' analysis revealed that this was due to disulfide bond mismatches and unstable protein structure during preparation. Mutant-DXd showed virtually no precipitation during preparation, indicating a minimal probability of disulfide bond mismatches and a more stable protein structure compared to antibodies 3-DXd and 4-DXd.

[0149] The DAR value of the antibody-drug conjugate was 8, as determined by hydrophobic interaction chromatography and LC-MS analysis.

[0150] By size exclusion chromatography analysis, the results are shown in Figure 7. After coupling, the peak shapes of mutants 6-DXd, mutant 20-DXd, mutant 21-DXd, mutant 24-DXd, mutant 25-DXd and mutant 26-DXd were broadened and poorly symmetrical, indicating that the structures of mutants 6, mutant 20, mutant 21, mutant 24, mutant 25 and mutant 26 became loose after coupling with small molecule drugs; the peak shapes of mutants 22-DXd, mutant 23-DXd, mutant 27-DXd and mutant 28-DXd after coupling were almost the same as before coupling, indicating that the structure of the antibody-drug conjugates obtained after coupling of mutants 22, mutant 23, mutant 27 and mutant 28 with small molecule drugs did not change significantly.

[0151] The above experiments show that inserting the acidic amino acid aspartic acid (D) or glutamic acid (E) between the cysteine ​​residues at positions 4 and 5 can not only significantly reduce the formation of heavy chain antibody aggregates, but also prepare heavy chain antibody-drug conjugates that can maintain their structural stability.

[0152] Example 5: Effect of inserting different numbers of amino acids into the mutation target region on antibody stability

[0153] As shown in Example 4, the insertion of acidic amino acid residues aspartic acid (D) and glutamic acid (E) between the cysteine ​​residues at positions 4 and 5 can not only reduce the aggregates of heavy chain antibodies, but also facilitate the preparation of antibody-drug conjugates. Further investigation was conducted to determine whether the insertion of a larger number of aspartic acid (D) or glutamic acid (E) between the cysteine ​​residues at positions 4 and 5 affects the stability of the antibody. Referring to Example 3, the insertion of 2, 4, 5, 6, and 7 amino acid residues of aspartic acid (D) or glutamic acid (E) into the corresponding regions of antibody 3 is used as an example, as shown in Table 6.

[0154] Table 6

[0155] The different mutants listed in Table 6 were expressed and purified, and size exclusion chromatography analysis results, as shown in Table 6, showed that none of the heavy chain antibodies containing the mutants formed aggregates. Dynamic light scattering (DLS) was then used to test the thermal stability of these 10 heavy chain antibodies. The DLS (Wyatt) instrument measures light scattering when the antibody is heated from 25°C to 85°C, thereby obtaining the aggregation temperature (Tag) of the antibody.

[0156] As shown in Table 6, as the number of aspartic acid (D) or glutamic acid (E) insertions increases, the polymerization temperature Tagg value of the heavy chain antibody gradually decreases. Therefore, between the cysteine ​​residues at positions 4 and 5, as the number of acidic amino acids aspartic acid (D) or glutamic acid (E) inserted increases, the stability of the heavy chain antibody will deteriorate. When two acidic amino acids aspartic acid (D) or glutamic acid (E) are inserted, the stability is the best.

[0157] Example 6: Mutating human IgG2 or IgG4 Fc to construct heavy chain antibodies

[0158] To investigate whether the mutational modification could be applied to human IgG2 Fc or IgG4 Fc, without affecting antibody production and increasing the number of antibody-conjugated small molecules, the modified mutation region (using the insertion of two glutamic acid residues (E) or two aspartic acid residues (D) as an example) was replaced with the corresponding region of the IgG2 and IgG4 Fc. The resulting heavy chain antibodies were named Mutant 37, Mutant 38, Mutant 39, and Mutant 40, respectively, as shown in Table 7.

[0159] The amino acid sequence of human IgG2 Fc is as follows (SEQ ID NO: 4):

[0160] The amino acid sequence of human IgG4 Fc is as follows (SEQ ID NO: 5):

[0161] Table 7

[0162] VHH1 was constructed into heavy chain antibodies with human IgG2 or IgG4 Fc modified as shown in Table 7. After expression and purification, mutants 37, 38, 39, and 40 were obtained. As shown in the SDS-PAGE of Figure 8 and the size exclusion chromatography analysis of Table 7, after the modified human IgG2 or IgG4 Fc was linked to the antibody, the obtained mutants produced almost no aggregates.

[0163] Mutant 37, mutant 38, mutant 39 and mutant 40 were prepared into antibody-drug conjugates, referring to Example 4, to obtain mutant 37-DXd, mutant 38-DXd, mutant 39-DXd and mutant 40-DXd, respectively.

[0164] Hydrophobic interaction chromatography and LC-MS analysis showed that the DAR values ​​of mutants 37-DXd, 38-DXd, 39-DXd and 40-DXd were 8.

[0165] Size exclusion chromatography analysis was performed, and the results are shown in Figure 9. The chromatographic peak shapes of mutants 37-DXd, mutant 38-DXd, mutant 39-DXd, and mutant 40-DXd were normal, indicating that after modifying human IgG2 or IgG4, the heavy chain antibodies prepared not only do not produce aggregates, but also can increase the number of antibody-coupled small molecules.

[0166] Example 7: Effect of mutated heavy chain antibodies on cell binding ability

[0167] Next, the present inventors verified whether the cell binding ability of the mutated heavy chain antibody was different from that of the wild-type Fc.

[0168] Because VHH1 can bind to human ovarian cancer cells OVCAR-3, flow cytometry was used to incubate antibody 1, mutant 22, mutant 23, mutant 37, mutant 38, mutant 39, mutant 40, antibody 1-DXd, mutant 22-DXd, mutant 23-DXd, mutant 37-DXd, mutant 38-DXd, mutant 39-DXd, and mutant 40-DXd with human ovarian cancer cells OVCAR-3, then wash away the unbound antibody, and then add a secondary antibody with a PE-labeled anti-human Fc antibody. After the incubation, wash away the unbound secondary antibody and detect by flow cytometry. As shown in Figure 10, the binding ability of the heavy chain antibody composed of VHH1 and mutants of human IgG1, IgG2 and IgG4 Fc to OVCAR-3 cells is the same as that of the heavy chain antibody constructed with wild-type Fc. Moreover, compared with VHH1-IgG1-DXd coupled with 4 small molecule drugs, the binding ability to OVCAR-3 cells is not affected after coupling with 8 small molecule drugs, indicating that the mutation does not affect the affinity of the heavy chain antibody to the antigen.

[0169] Example 8. Preparation of Antibody-Drug Conjugates Using Heavy Chain Antibodies Prepared After Mutation and Their Efficacy

[0170] OVCAR-3 cells were seeded at 5,000 cells per well in a 96-well plate and cultured overnight at 37°C. Antibodies 1-DXd, mutant 22-DXd, mutant 23-DXd, mutant 37-DXd, mutant 38-DXd, mutant 39-DXd, and mutant 40-DXd were serially diluted and added to the 96-well plate containing OVCAR-3 cells. The cells were cultured at 37°C for 5 days. Cell viability was assessed using CellTiter-Glo (Promega) to assess the cell-killing efficacy of the antibody-drug conjugate.

[0171] The results are shown in Figure 11. Compared with antibody 1-DXd conjugated with four small molecule drugs, mutants 22-DXd, 23-DXd, 37-DXd, 38-DXd, 39-DXd and 40-DXd caused significant killing of OVCAR-3 cells. According to the concentration of DXd, the IC50 can be increased by 7 to 20 times, indicating that the antibody-drug conjugates of the modified heavy chain antibody are significantly more effective in killing tumor cells than the antibody-drug conjugates of the heavy chain antibody constructed using the Fc of wild-type human IgG1.

[0172] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for increasing the functional molecular loading of an antigen binding fragment-human IgG Fc fusion protein and reducing the generation of polymers after expression, characterized in that: The method comprises: (1) Providing an antigen-binding fragment-human IgG Fc fusion protein, wherein the N-terminal sequence of the human IgG Fc is a modified sequence, wherein the modification is: replacing the 6-15 amino acid residues at the N-terminus with the amino acid residues shown in SEQ ID NO: 6, and performing a modification selected from the following group based on the sequence: (a) inserting 1 to 9 amino acid residues between, before or after the CC residues at positions 4 and 5 of SEQ ID NO: 6; (b) Mutating any one of the 4th and 5th Cs of SEQ ID NO:

6.

2. The method according to claim 1, characterized in that In (a), 2-8 non-cysteine ​​residues are inserted between the two CC residues.

3. The method according to claim 1, characterized in that The amino acid residues for insertion or mutation modification include aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, histidine, or a combination thereof.

4. The method according to claim 3, characterized in that The amino acid residues used for insertion are acidic amino acids, including aspartic acid, glutamic acid, or a combination thereof.

5. The method according to claim 1, characterized in that The method further comprises: (2) connecting functional molecules to the antigen binding fragment-human IgG Fc fusion protein; the loading amount of the functional molecules is 6-8.

6. The method according to claim 1, characterized in that The functional molecules include: small molecule anti-tumor drugs, cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or combinations thereof.

7. The method according to claim 6, characterized in that The molecule targeting the tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; or The tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin; or The detectable markers include: fluorescent markers and colorimetric markers.

8. The method according to claim 7, characterized in that The anti-tumor toxins include: toxins acting on tubulin; toxins acting on DNA; compounds acting on intracellular metabolism, transcription, translation or signal transduction; or The anti-tumor cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof; or The antibody that binds to tumor surface markers is an antibody that recognizes tumor antigens.

9. The method according to claim 8, characterized in that The toxin acting on tubulin is monomethyl auristatin, its related compounds or derivatives thereof; or The toxin acting on tubulin is maytansinoid, its related compounds or its derivatives; or The toxins acting on DNA are duocarmycin, calicheamicin, pyrrolobenzodiazepines, SN-38, DXd, related compounds or derivatives thereof.

10. The method according to claim 1, characterized in that The human IgG is selected from: human IgG1, human IgG2 or human IgG4; or The antigen-binding fragment includes: a polypeptide capable of binding to an antigen in a heavy chain antibody, a single chain antibody, a single domain antibody, a BiTE antibody, a DART antibody, a Fv antibody, or a Fd antibody; or The antigen-binding fragment is the variable region of the heavy chain or light chain of an antibody, or a combination of the two.

11. A modified human IgG Fc variant, wherein the N-terminal sequence of the human IgG Fc is a modified sequence, wherein the N-terminal 6-15 amino acid residues are replaced with the amino acid residues shown in SEQ ID NO: 6, and based on the sequence, a modification selected from the following group is performed: (a) inserting 1 to 9 amino acid residues between, before or after the CC residues at positions 4 and 5 of SEQ ID NO: 6; (b) Mutating any one of the 4th and 5th Cs of SEQ ID NO:

6.

12. The engineered human IgG Fc variant according to claim 11, wherein In (a), 2-8 amino acid residues are inserted between the CC residues.

13. The engineered human IgG Fc variant of claim 11, wherein The amino acid residues for insertion or mutation modification include aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, histidine, or a combination thereof.

14. The engineered human IgG Fc variant of claim 11, wherein The amino acid residues used for insertion are acidic amino acids, including aspartic acid, glutamic acid, or a combination thereof.

15. The engineered human IgG Fc variant of claim 11, wherein The N-terminal sequence of the human IgG Fc is a modified sequence, and the modification includes: mutating the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, and SEQ ID NO:

7.

16. Use of the modified human IgG Fc variant according to any one of claims 11 to 15 for preparing an antigen binding fragment-human IgG Fc fusion protein; when a functional molecule is linked to the antigen binding fragment-human IgG Fc fusion protein, the functional molecule loading amount is 6-8.

17. A fusion protein, which is an antigen binding fragment-human IgG Fc fusion protein; wherein the human IgG Fc is the human IgG Fc variant according to any one of claims 11 to 15.

18. A polynucleotide encoding the human IgG Fc variant according to any one of claims 11 to 15, or encoding the fusion protein according to claim 17.

19. An expression construct comprising the polynucleotide of claim 18.

20. An expression system comprising the expression construct of claim 19 or the polynucleotide of claim 18 integrated into its genome.

21. A method for preparing the human IgG Fc variant according to any one of claims 11 to 15, or the fusion protein according to claim 17, comprising: Under conditions suitable for expression, the expression system of claim 20 is used for expression to obtain the human IgG Fc variant or fusion protein.

22. A conjugate comprising: The fusion protein of claim 17; and a functional molecule connected to the fusion protein; The loading amount of the functional molecules is 6-8.

23. The conjugate according to claim 22, characterized in that The functional molecules include: small molecule anti-tumor drugs, cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or combinations thereof.

24. A pharmaceutical composition or a kit, characterized in that: It includes: The fusion protein of claim 17, or the conjugate of any one of claims 22-23.

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