Method for preparing polypeptide conjugate

Abstract

Provided is a method for preparing a heterologous polypeptide conjugate by using a random and / or site-specific conjugation method in combination with in vitro antibody recombination technology. The heterologous polypeptide conjugate comprises single-target and dual-payload, double-target and single-payload and double-target and dual-payload polypeptide conjugates, and the order of payload conjugation and the order of in vitro recombination can be adjusted.

Description

A method for preparing a polypeptide conjugate

[0001] Related applications

[0002] This application claims priority and related interests in Chinese Patent Application No. 202510483550.1, filed on April 16, 2025; Chinese Patent Application No. 202511734905.6, filed on November 24, 2025; and Chinese Patent Application No. 202610475594.4, filed on April 10, 2026, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the fields of medicinal chemistry and biotechnology drugs, specifically to a method for preparing peptide conjugates using antibody engineering and conjugation techniques. Background Technology

[0004] Antibody drugs are currently widely used in the treatment of various diseases, including oncology, immunology, and anti-inflammation. Antibody drug conjugates (ADCs) are a class of emerging biotechnology drugs composed of antibodies, cytotoxins, and linkers. Currently, more than 10 ADC drugs have been approved for marketing worldwide.

[0005] Linkers and antibodies are coupled in specific ways. The most common coupling method is random coupling via naturally occurring lysine amino residues or cysteine ​​thiol residues. ADCs produced by this method typically exhibit poor homogeneity and unstable coupling sites. To address this issue, various site-directed coupling methods have emerged, including engineered cysteine ​​coupling methods, bridging methods, coupling methods based on non-natural amino acids, affinity peptide-labeled coupling methods, enzymatic coupling methods, glycosyl coupling methods, and other selective chemical coupling methods.

[0006] Currently, most ADC drugs in clinical development have single-target capabilities and carry a single type of payload. With several dual-target, single-load ADCs entering clinical trials and achieving preliminary clinical validation, dual-target, single-load ADCs have become a new trend in ADC development. Dual-target ADCs offer better indication coverage than single-target ADCs and can potentially reach more patients. The preparation of heterodimeric antibodies is fundamental to the preparation of dual-target ADCs. Technologies such as Fab exchange and "Knobs-into-Holes" can be used to prevent heavy chain mismatches, while technologies such as Crossmab and Duetmab can be used to prevent light chain mismatches.

[0007] Dual-payload ADCs (dpADCs) represent another innovative direction in ADC development. Since load resistance is a significant factor contributing to drug resistance in ADCs, a dual-payload design may enhance the efficacy of ADC drugs and slow the development of resistance. With advancements in conjugation technology, the uniform preparation of dual-payload ADCs has become feasible, and several molecules are already progressing through preclinical development.

[0008] Double-target and dual-payload ADCs (ddADCs) offer the potential to combine the advantages of dual targets and dual payloads, resulting in ADCs with broader tumor recognition, better efficacy, and lower resistance to drug resistance. However, due to the overall complexity of the design of double-target and dual-payload ADCs, existing technologies often produce ADCs with poor uniformity. Furthermore, the symmetrical approach typically used in current preparation methods makes fine-tuning the drug loading (DAR value) of specific payloads impractical. Currently, the research and clinical needs for double-target and dual-payload ADC preparation remain unmet. Summary of the Invention

[0009] This disclosure provides a method for preparing a highly uniform heterodimeric polypeptide conjugate with selectable drug loading, either single-target dual-load, dual-target single-load, or dual-target dual-load, from a polypeptide conjugate or a conjugated polypeptide.

[0010] This disclosure provides a method for preparing heterodimeric polypeptide conjugates, the method comprising the following steps:

[0011] a) Provide: ① a first polypeptide conjugate, or a first polypeptide modified by conjugation, and ② a second polypeptide conjugate, or a second polypeptide modified by conjugation, or a second polypeptide;

[0012] The first polypeptide contains a first Fc region, and the second polypeptide contains a second Fc region. The first polypeptide conjugate is coupled to the first polypeptide containing the first Fc region, and the second polypeptide conjugate is coupled to the second polypeptide containing the second Fc region. The first polypeptide modified by coupling is modified to have a different coupling position with the first polypeptide, and the second polypeptide modified by coupling is modified to have a different coupling position with the second polypeptide.

[0013] The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region, that is, stronger than the homodimer interaction formed by the first Fc region itself or the homodimer interaction formed by the second Fc region itself.

[0014] b) Co-incubation in the same reaction system of: ① a first polypeptide conjugate, or a first polypeptide modified by coupling, and ② a second polypeptide conjugate, or a second polypeptide modified by coupling, or a second polypeptide; and,

[0015] c) Obtain the heterodimeric polypeptide conjugate.

[0016] In some implementations, after incubation of the first and second peptides modified by coupling, a load is added to carry out a coupling reaction to obtain heterodimeric peptide conjugates.

[0017] In some embodiments, the preparation method further includes: firstly separating the first polypeptide conjugate containing the first Fc region or the first polypeptide modified by coupling and the second polypeptide conjugate containing the second Fc region, the second polypeptide modified by coupling, or the second polypeptide from their respective homodimers; after the homodimers are separated, since the heterodimer interaction between the first Fc region and the second Fc region is stronger than the homodimer interaction between the first Fc region and the second Fc region, the first Fc region and the second Fc region preferentially pair up and reassemble to form a heterodimeric polypeptide conjugate.

[0018] In some preferred embodiments, the first and second peptides used to prepare heterodimeric peptide conjugates do not need to undergo hinge region disulfide bond isomerization.

[0019] In some preferred embodiments, the first and second polypeptides used to prepare the heterodimeric polypeptide conjugates require disulfide bond isomerization in the hinge region. Methods for separating the homodimers include providing reducing conditions to open the disulfide bonds in the first and second Fc regions, followed by providing oxidizing conditions to reassemble the heterodimers.

[0020] In a first aspect, this disclosure provides a method for preparing a heterodimeric polypeptide conjugate, the method comprising the following steps:

[0021] a) Provide: ① a first polypeptide conjugate, and ② a second polypeptide conjugate, or a second polypeptide modified by conjugation, or a second polypeptide;

[0022] The first polypeptide contains a first Fc region, and the second polypeptide contains a second Fc region. The first polypeptide conjugate is coupled to the first polypeptide containing the first Fc region, and the second polypeptide conjugate is coupled to the second polypeptide containing the second Fc region. The second polypeptide modified by coupling is modified to have a different coupling position with the second polypeptide.

[0023] The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region respectively.

[0024] b) Co-incubation in the same reaction system of: ① a first polypeptide conjugate, and ② a second polypeptide conjugate, or a second polypeptide modified by conjugation, or a second polypeptide; and,

[0025] c) Obtain the heterodimeric polypeptide conjugate.

[0026] In some embodiments, after incubating the first polypeptide conjugate and the coupled second polypeptide in steps b) and c) to obtain a heterodimeric polypeptide conjugate, the preparation method further includes step d): adding a load to cause the coupled second polypeptide contained in the heterodimeric polypeptide conjugate to undergo a coupling reaction with the load, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load.

[0027] In some embodiments, after incubating the first polypeptide conjugate and the second polypeptide in steps b) and c) to obtain a heterodimeric polypeptide conjugate, the preparation method further includes step d): adding a load to cause the second polypeptide contained in the heterodimeric polypeptide conjugate to undergo a coupling reaction with the load, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load.

[0028] In some embodiments, after incubating the first polypeptide conjugate and the second polypeptide in steps b) and c) to obtain a heterodimeric polypeptide conjugate, the preparation method further includes step d): further modifying the second polypeptide in the heterodimeric polypeptide conjugate into a coupled second polypeptide, and then adding a load to cause the coupled second polypeptide contained in the heterodimeric polypeptide conjugate and the load to undergo a coupling reaction, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0029] Secondly, this disclosure provides a method for preparing heterodimeric polypeptide conjugates, the method comprising the following steps:

[0030] a) Provide: ① a first polypeptide modified by coupling, and ② a second polypeptide modified by coupling, or a second polypeptide;

[0031] The first polypeptide contains a first Fc region, and the second polypeptide contains a second Fc region. The first polypeptide, which has been coupled and modified, contains a first Fc region and is modified to have a different coupling position with the first polypeptide. The second polypeptide, which has been coupled and modified, contains a second Fc region and is modified to have a different coupling position with the second polypeptide.

[0032] The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region respectively.

[0033] b) Co-incubate in the same reaction system: ① the first polypeptide modified by coupling, and ② the second polypeptide modified by coupling, or the second polypeptide;

[0034] c-1) Obtain the coupled-modified heterodimeric polypeptide;

[0035] c-2) Add a load to cause it to undergo a coupling reaction with the first peptide and / or the second peptide contained in the coupled-modified heterodimeric polypeptide, thereby obtaining a heterodimeric polypeptide conjugate with a first peptide coupled load and / or a second peptide coupled load.

[0036] In some preferred embodiments, after incubating the first and second peptides modified by coupling in step b) and obtaining the heterodimeric peptide modified by coupling in step c-1), the second peptide contained in the heterodimeric peptide is further modified into a second peptide modified by coupling, and then the coupling reaction in step c-2) is carried out.

[0037] In some preferred embodiments, after incubating the first and second peptides modified by coupling in step b), obtaining the modified heterodimeric peptide in step c-1), and adding a load in step c-2) to couple the first peptide contained in the modified heterodimeric peptide to obtain a heterodimeric conjugate, the second peptide in the heterodimeric peptide is further modified into a second peptide modified by coupling.

[0038] In some preferred embodiments, after step c-2) adding a load to couple with the second polypeptide contained in the coupled-modified heterodimeric polypeptide or the coupled-modified second polypeptide, the method further includes step d): adding a load to couple with the coupled-modified first polypeptide contained in the heterodimeric polypeptide conjugate to obtain a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0039] In some preferred embodiments, after step c-2) adding a load to couple with the first polypeptide included in the coupled-modified heterodimeric polypeptide to obtain a heterodimeric polypeptide conjugate, the method further includes step d): adding a load to couple with the second polypeptide included in the heterodimeric polypeptide conjugate or the second polypeptide coupled to obtain a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0040] In some preferred embodiments, step c-2) involves adding a load to couple it with the first polypeptide in the coupled heterodimeric polypeptide to obtain a heterodimeric polypeptide conjugate. After further modifying the second polypeptide in the heterodimeric polypeptide conjugate into a coupled second polypeptide, the method further includes the following steps:

[0041] d) Add a load to cause it to couple with the modified second polypeptide contained in the heterodimeric polypeptide conjugate, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0042] In some embodiments, in step c-2), a load is added so that it simultaneously couples with the first and second peptides, or the second peptide, contained in the coupled heterodimeric polypeptide, thereby obtaining a heterodimeric polypeptide conjugate in which both the first and second peptides are coupled with loads.

[0043] In some embodiments, the first polypeptide is a first antibody further containing a first antigen / target binding fragment, or a first fusion protein containing a first Fc region, or containing a first Fc region; and / or, the second polypeptide is a second antibody further containing a second antigen / target binding fragment, or a second fusion protein containing a second Fc region, or containing a second Fc region. In some preferred embodiments, the first polypeptide and the second polypeptide bind to the same epitope of the same target, or different epitopes of the same target, or epitopes of different targets.

[0044] In some embodiments, the target bound by the first and / or second polypeptide is selected from any of the following antigens or any combination thereof: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Big3, SPARC, BCR, CD79, CD37, EGFrRvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, MRP3, TROP2, Nectin-4, CLDN 18.2, CLDN6, CDH6, B7-H3, ROR1, PSMA, PD-L1, FRα, DLL3, CDH17, 5T4, and Tissue. Factor, PTK7, BCMA, NaPi2b, B7-H4, LIV-1, CEACAM5, CD22, CD33, MSLN, CD30, GPC3, IL3RA, FGFR2b, CDCP1, CD73, uPARAP, KIT, IL2RA, ICAM-1, GUCY2C, CDH3, CD79b, CD70, VEGFR2, VEGF, TFR1, S TEAP1, SEZ6, NCAM1, MUC16, LRRC15, IL13RA2, DLK1, CLL-1, CD45, CD37, BCL2L1, KIF11, ITGB6, IL1RAP, FLT3, FGFR2, EphA2, DPP-4, CD7, CD47, TRAILR2, TLR7, Tau, Tumor-associated carbohydrate antigen (Tumor-associated carbohydrate (antigen), SORT1, SLC1A5, SDC1, PD-1, NMT, NA+ / K+ATPASE, LY6E, IGF-1R, HSP70, glucocorticoid receptor (GR), FGFR3, ENPP3, CD105, CTLA4, CLDN1, CEACAM6, CD228, CD147, ADAM9, AMHR2, ALPP, ALPG or LY6G6D, etc.; and / or, CD3, CD4, CD8, CD40, CD25, CD28, CD16, CD89, CD32, CD64, FcεRI or CD1, etc.

[0045] In some embodiments, the first Fc region is selected from IgG1, IgG2, IgG3, and IgG4 isotypes, and / or, the second Fc region is selected from IgG1, IgG2, IgG3, and IgG4 isotypes. In some preferred embodiments, both the first Fc region and the second Fc region are IgG1 or IgG4 isotypes.

[0046] In some embodiments, the first Fc region and / or the second Fc region contains amino acid substitutions, wherein the amino acid substitutions are one or more charge-based amino acid substitutions and / or non-electrostatic-based amino acid substitutions in the first Fc region and / or the second Fc region.

[0047] In some preferred embodiments, the one or more charge-based amino acid substitutions refer to the first Fc region and / or the second Fc region containing one or more positively charged amino acids and negatively charged amino acids, or positively charged amino acids and uncharged amino acids, or negatively charged amino acids and uncharged amino acids.

[0048] In some preferred embodiments, the first Fc region and the second Fc region each contain amino acid substitutions selected from any of the following groups, designated by EU numbers: T366S / L368A / Y407V, T366W; S354C / T366W, Y349C / T366S / L368A / Y407V; T366Y, Y407T; T366W, Y407A; T394W, F405A; T366Y / F405A, T394W / Y407T; T366W / F405W, T394S / Y407A; F405W, T394S; D399C, K392C; T366W / D399C, T366S / L368 A / K392C / Y407V; T366W / K392C, T366S / L368A / D399C / Y407V; S354C / T366W , Y349C / T366S / L368A / Y407V; Y349C / T366W, S354C / T366S / L368A / Y407V; E356C / T366W, Y349C / T366S / L368A / Y407V; Y349C / T366W, E356C / T366S / L 368A / Y407V; E357C / T366W, Y349C / T366S / L368A / Y407V; Y349C / T366W, E3 57C / T366S / L368A / Y407V; T366K, L351D; T366K, Y349E / D; T366K, L368E / D ;T366K, L351D / Y349D / R355D; T366K, Y349E / D / L368E / D / L351D; T366K / L3 51K, L351D; T366K / L351K, Y349E / D; T366K / L351K, L368E / D; T366K / L351K , Y349E / D / L368E / D / L351D; T366K / L351K, L351D / Y349D / R355D; L351K, L3 51D; K370E / D399K / K439D, D356K / E357K / K409D; K409D, D399K; K409E, D39 9K; K409E, D399R; K409D, D399R; D339K, E356K; E356K / D399K, K392D / K409 D; E356K, K439E; E356D, K439R; E356K / D399K, K409D / K439D; E357K, K370D ;E357K / D399K, K370D / K409D; E356K / E357K / D399K, K370D / K392D / K409D;E356K / E357K / D399K,K370E / K392D / K409D;E357K / D399K,K392D / K409D;D399K / R,K392D;K392D / K409D,D399K;K360D / K409D,D399K;S364R / D399K,K370S / Y349T / K409D;S364R / D399K / E357N / T350V,K370S / Y349T / Q347D / K409D / T350V;L351Y / S400E / F405A / Y407V / T350V,T366L / N390R / K392M / T394W / T350V;K409E / T411R,D399K / K370T;S364R / D399K / E357N,K370S / Y349T / Q347D / K409D;S364R / D399K,K370S / Y349T / Q347D / K409D;F405L,K409R;WT,F405L / R409K;T350I / K370T / F405L,K409R;K370W,K409R;D399A / F / G / H / I / L / M / N / R / S / T / V / W / Y,K409R;T366A / D / E / F / G / H / I / L / M / Q / V / Y,K409R;L368A / D / E / G / H / N / R / S / T / V / Q,K409A / G / R / H;D399F / H / K / R / Q,K409A / G / R / H;F405I / K / L / S / T / V / W,K409A / G / R / H;Y407L / W / Q,K409A / G / R / H;D360K,K402D;D403K,K419D;E357K,K370E;K249E / K288E,E236K / D278K;H435R,WT;S364H,Y349T;S364H / F405A,Y349T / T394F;S364H,Y349T;D399R / S364H,K409D / Y349T / K370S;D399K / S364H,K409D / Y349T / K370S;D399K / E356K,K370D / K409D;S364K / N390P / S400K,S364D / K370D / N390D / K392G / S400D。;

[0049] In some preferred embodiments, the first Fc region and the second Fc region each contain amino acid substitutions selected from any one of the following groups, wherein the substitutions are numbered EU: D399R / S364H, K409D / Y349T / K370S; D399K / S364R, K409D / Y349T / K370S; D399K / S364H, K409D / Y349T / K370S; D399K / E356K, K392D / K409D; D399K / E 356K, K370D / K409D; S364K / N390P / S400K, S364D / K370D / N390D / K392G / S400D; D399K / E357K, K392D / K409D ;D399K / E357K, K370D / K409D; F405L, K409R; WT, F405L / R409K; T366S / L368A / Y407V / Y349C, T366W / S354C.

[0050] In some embodiments, the first polypeptide comprises a first heavy chain H1 and a first light chain L1; and / or, the second polypeptide comprises a second heavy chain H2 and a second light chain L2; wherein H1 and H2 comprise CH1 domains, and L1 and L2 comprise CL domains.

[0051] In some preferred embodiments, the first polypeptide and / or the second polypeptide improve the correct pairing of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 using techniques such as CrossMab, DuetMab, common light chain, and common heavy chain. In some preferred embodiments, the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 comprise: (a) a modified CH1 domain, wherein the modified CH1 domain comprises a substitution of a native non-cysteine ​​amino acid for a cysteine ​​residue; and (b) a modified corresponding CL domain, wherein the modified CL domain comprises a substitution of a native non-cysteine ​​amino acid for a cysteine ​​residue. In some preferred embodiments, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following substitutions, wherein the substitutions are designated by EU numbers: CH1: the amino acid at position 126 is substituted with cysteine, and the cysteine ​​at position 220 is substituted with a non-cysteine ​​amino acid; CL: the amino acid at position 121 is substituted with cysteine, and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid. In a more preferred embodiment, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following substitutions: CH1: F126C / C220V, CL: S121C / C214V.

[0052] In some embodiments, the first polypeptide is conjugated to one or more types of payloads, each type containing one or more payloads. In some embodiments, the second polypeptide is conjugated to one or more types of payloads, each type containing one or more payloads. In some embodiments, the type and / or number of payloads conjugated to the first polypeptide and the second polypeptide may be exactly the same, partially the same, or different.

[0053] In some embodiments, the payload is a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a peptide, a nucleic acid and related drugs, a radiolabeled isotope, a contrast agent, or a magnetic resonance imaging agent; the small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, DXd, exatecan, docarmycin, amanitin, PBDs, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of the above compounds, or the small molecule drug is a radiotherapy agent. In some preferred embodiments, the first peptide and the second peptide are coupled with different types of payloads.

[0054] In some embodiments, the first polypeptide is conjugated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 payloads; and / or, the second polypeptide is conjugated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 payloads. In some preferred embodiments, the first polypeptide is conjugated with 1, 2, 3, 4, 5, or 6 payloads; and / or, the second polypeptide is conjugated with 1, 2, 3, 4, 5, or 6 payloads. In some preferred embodiments, the first polypeptide and the second polypeptide are conjugated with different numbers of payloads.

[0055] In some embodiments, the first peptide-coupled payload or the second peptide-coupled payload is prepared via fusion expression, random coupling, or site-specific coupling. In some embodiments, the preparation method of the first peptide-coupled payload and / or the second peptide-coupled payload can be a combination of one or more coupling methods, and the preparation methods of the first peptide-coupled payload and the second peptide-coupled payload can be the same, partially the same, or different. In some embodiments, the payload is coupled to the same site, partially the same site, or different sites of the first peptide and the second peptide.

[0056] In some preferred embodiments, the random coupling method is a lysine-based random coupling method, a cysteine-based random coupling method, or a bridging-based method. In some more preferred embodiments, the random coupling method is a cysteine-based coupling method or a bridging-based method.

[0057] In some preferred embodiments, the site-directed coupling method is a site-directed coupling method based on non-natural amino acids, a site-directed coupling method based on engineered cysteine ​​mutant antibodies, an enzyme-based site-directed coupling method, a site-directed coupling method based on Fc affinity peptides, or a sugar-based site-directed coupling method. In some more preferred embodiments, the site-directed coupling method is an enzyme-based site-directed coupling method or a sugar-based site-directed coupling method.

[0058] In some preferred embodiments, the enzyme-based site-directed coupling method is selected from coupling methods based on the following enzymes and their mutants, or modified enzymes containing their catalytic domains: sortase, prenyltransferase, formylglycine-generating enzyme, transglutaminase, spyLigase, horseradish peroxidase, tyrosinase, phosphopantetheinyl transferase, etc. In some more preferred embodiments, the transglutaminase is selected from microbial transglutaminase (mTGase) and its mutants, or modified enzymes containing its catalytic domain.

[0059] In some preferred embodiments, the sugar-based site-directed coupling method is selected from glycosylation-based coupling methods, or coupling methods based on enzymes and their mutants, or modified enzymes containing their catalytic domains: galactosyltransferase (GalT), beta-N-acetylglucosaminidase (ENGase), α-L-fucosidase, etc. In some preferred embodiments, the galactosyltransferase (GalT) is selected from β(1,4)-Gal-Tl and its mutants, or modified enzymes containing its catalytic domain. In some preferred embodiments, the beta-N-acetylglucosaminidase (ENGase) is selected from Endo Si, Endo Se2, Endo S, Endo S2, Endo F3, Endo-M and their mutants, or modified enzymes containing their catalytic domain.

[0060] In some preferred embodiments, the first polypeptide and the second polypeptide are coupled independently to the load via a method selected from disaccharide-linked site-specific coupling, random coupling, and / or bridging coupling.

[0061] In some embodiments, the first and / or second polypeptides couple the loading to a conserved cysteine ​​residue of the polypeptide via a thiol-reactive group such as maleimide, 2-methylsulfonyl pyrimidine, or haloacetamide. In some preferred embodiments, the linker is a bridging linker.

[0062] In some embodiments, the preparation method includes mixing and incubating a first polypeptide and / or a second polypeptide with a reducing agent, adding a loading agent to react, and purifying to obtain a first polypeptide conjugate and / or a second polypeptide conjugate.

[0063] In some preferred embodiments, the preparation method includes adding an organic solvent.

[0064] In some preferred embodiments, the preparation method includes adding a quencher to terminate the reaction.

[0065] In some embodiments, the first polypeptide and / or the second polypeptide are enzymatically coupled to the enzymatic reaction site on the polypeptide via a linker. In some preferred embodiments, the linker is a disaccharide linker or a complex glycan linker, and the load is coupled at the conserved N297 N-glycosylation modification site.

[0066] In some embodiments, the preparation method includes the step of conjugating a first polypeptide and / or a second polypeptide via a disaccharide linker, an ENGase, and a load; for example, it includes mixing the first polypeptide and the second polypeptide with a disaccharide linker and an ENGase connected with a load, respectively, and incubating them to obtain the first polypeptide conjugate and the second polypeptide conjugate, respectively.

[0067] In some preferred embodiments, the first polypeptide and the second polypeptide are respectively mixed with a disaccharide linker and ENGase, and incubated to obtain the desired non-naturally glycoengineered first polypeptide or non-naturally glycoengineered second polypeptide. Optionally, the non-naturally glycoengineered first polypeptide and the non-naturally glycoengineered second polypeptide are then respectively mixed with a load, and incubated to obtain a first polypeptide conjugate or a second polypeptide conjugate.

[0068] In some preferred embodiments, incubating the first polypeptide conjugate and the second polypeptide conjugate yields a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0069] In some preferred embodiments, after incubating the first polypeptide conjugate and the non-natural sugar engineered second polypeptide, or obtaining a heterodimeric polypeptide conjugate from the second polypeptide, the heterodimeric polypeptide conjugate and the load are mixed, and after incubation, a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load is obtained.

[0070] In some preferred embodiments, after incubating the first polypeptide conjugate and the second polypeptide to obtain a heterodimeric polypeptide conjugate, the heterodimeric polypeptide conjugate is mixed with a disaccharide linker and ENGase, and incubated to obtain a non-natural sugar-engineered heterodimeric polypeptide conjugate modified with the second polypeptide. Optionally, the non-natural sugar-engineered heterodimeric polypeptide conjugate modified with the second polypeptide is then mixed with a loading, and incubated to obtain a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the loading.

[0071] In some embodiments, the preparation method includes the step of coupling a first polypeptide and / or a second polypeptide via a disaccharide linker, an ENGase, and a load; for example, it includes mixing the first polypeptide and the second polypeptide with a disaccharide linker and an ENGase linked with a load, respectively, and incubating them to obtain a non-natural sugar engineered first polypeptide or a non-natural sugar engineered second polypeptide.

[0072] In some preferred embodiments, a non-naturally glycoengineered first polypeptide and a non-naturally glycoengineered second polypeptide are incubated to obtain a non-naturally glycoengineered heterodimeric polypeptide in which both the first and second polypeptides are modified. Optionally, the non-naturally glycoengineered heterodimeric polypeptide and the loading are then mixed and incubated to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled with the loading.

[0073] In some preferred embodiments, a non-natural sugar engineered first polypeptide and a second polypeptide are incubated to obtain a non-natural sugar engineered heterodimeric polypeptide modified from the first polypeptide.

[0074] In some preferred embodiments, a non-natural sugar-engineered heterodimeric peptide modified with the first peptide is mixed with a load, and after incubation, a heterodimeric peptide conjugate in which both the first peptide and the second peptide are coupled with the load is obtained.

[0075] In some preferred embodiments, a non-natural sugar-engineered heterodimeric polypeptide modified with the first polypeptide is mixed with a disaccharide linker and ENGase, and incubated to obtain a non-natural sugar-engineered heterodimeric polypeptide modified with both the first polypeptide and the second polypeptide; optionally, the non-natural sugar-engineered heterodimeric polypeptide is then mixed with a load, and incubated to obtain a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load.

[0076] In some preferred embodiments, a non-natural sugar-engineered heterodimeric peptide modified with the first polypeptide is mixed with a load and incubated to obtain a heterodimeric peptide conjugate with the second polypeptide coupled with the load; optionally, the heterodimeric peptide conjugate is then mixed with the load and incubated to obtain a heterodimeric peptide conjugate with both the first polypeptide and the second polypeptide coupled with the load.

[0077] In some preferred embodiments, a non-natural sugar-engineered heterodimeric peptide modified with the first polypeptide is mixed with a load and incubated to obtain a heterodimeric polypeptide conjugate with the first polypeptide coupled to the load; optionally, the heterodimeric polypeptide conjugate is then mixed with the load and incubated to obtain a heterodimeric polypeptide conjugate with both the first polypeptide and the second polypeptide coupled to the load.

[0078] In some preferred embodiments, a non-natural sugar-engineered heterodimeric peptide modified with the first polypeptide is mixed with a load, and incubated to obtain a heterodimeric peptide conjugate with the first polypeptide coupled with the load; optionally, the heterodimeric peptide conjugate with the first polypeptide coupled with the load is then mixed with a disaccharide linker and ENGase, and incubated to obtain a heterodimeric peptide conjugate with the second polypeptide modified; optionally, the heterodimeric peptide conjugate is then mixed with a load, and incubated to obtain a heterodimeric peptide conjugate with both the first polypeptide and the second polypeptide coupled with the load.

[0079] In some embodiments, step b) of the preparation method further includes incubating the first polypeptide conjugate with the second polypeptide or a second polypeptide conjugate under reducing conditions sufficient to allow disulfide isomerization of cysteine ​​in the hinge region. In some preferred embodiments, the reducing conditions include the addition of a reducing agent selected from: 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine or a chemical derivative thereof, dithioerythritol, glutathione, L-cysteine, and β-mercaptoethanol. In some preferred embodiments, step b) includes incubation at a temperature of at least 20°C for at least 30 minutes in the presence of 2-mercaptoethylamine, dithiothreitol, or tris(2-carboxyethyl)phosphine or a chemical derivative thereof.

[0080] In some embodiments, step c) or step c-1) of the preparation method further includes removing the reducing agent.

[0081] In some embodiments, step c) or step c-1) of the preparation scheme further includes natural oxidation or the addition of an oxidizing agent.

[0082] In some embodiments, step c) or step c-1) further includes a purification step to remove the homodimer from the reaction system and purify to obtain the heterodimer polypeptide conjugate or heterodimer polypeptide.

[0083] In some embodiments, the preparation method includes the following steps:

[0084] a1) Prepare the first polypeptide and the second polypeptide respectively: Construct expression vectors containing the first polypeptide and the second polypeptide respectively, transform the expression vectors into host cells for expression, and purify to obtain the first polypeptide and the second polypeptide, wherein the first Fc region of the first polypeptide and the second Fc region of the second polypeptide contain different amino acid substitutions;

[0085] a2) Preparation of the first polypeptide conjugate and the second polypeptide conjugate: The first polypeptide conjugate and / or the second polypeptide conjugate are obtained by methods such as glycoconjugation, random cysteine ​​conjugation, or bridging, wherein,

[0086] The steps of the sugar coupling preparation method are as follows:

[0087] a2-1) Preparation of non-natural glycoengineered first polypeptide and / or non-natural glycoengineered second polypeptide: Mix the first polypeptide with the disaccharide linker and the glycoside endonuclease ENGase, and / or mix the second polypeptide with the disaccharide linker and the glycoside endonuclease ENGase, and purify after incubation to obtain the non-natural glycoengineered first polypeptide and the non-natural glycoengineered second polypeptide.

[0088] a2-2) Preparation of the first peptide conjugate and / or the second peptide conjugate: Add a non-natural sugar engineered first peptide and a linker-loader to a reaction system, and / or add a non-natural sugar engineered second peptide and a linker-loader to another reaction system, incubate them separately, confirm the reaction products by LC-MS, and obtain the first peptide conjugate and the second peptide conjugate.

[0089] The steps of the method for preparing cysteine ​​by random coupling or bridging are as follows:

[0090] a2-1) Mix the first polypeptide with a reducing agent, incubate, add the loading reaction, and purify to obtain the first polypeptide conjugate; and / or, in another reaction system, mix the second polypeptide with a reducing agent, incubate, add the loading reaction, and purify to obtain the second polypeptide conjugate.

[0091] b) Mix the first polypeptide conjugate and the second polypeptide conjugate in the same reaction system, add a reducing agent, and incubate;

[0092] c) Remove the reducing agent, allow natural oxidation, and purify to obtain heterodimeric polypeptide conjugates;

[0093] In step b), the first polypeptide conjugate and the second polypeptide conjugate are mixed in a molar ratio of 1:1, the concentration of the reducing agent is in the range of 0.5-100mM, the pH of the reaction system is 6-8, the temperature is 20-40℃, and the time is 0.5-48h.

[0094] In some embodiments, the preparation method includes the following steps:

[0095] a1) Prepare the first polypeptide and the second polypeptide respectively: Construct expression vectors containing the first polypeptide and the second polypeptide respectively, transform the expression vectors into host cells for expression, and purify to obtain the first polypeptide and the second polypeptide, wherein the first Fc region of the first polypeptide and the second Fc region of the second polypeptide contain different amino acid substitutions;

[0096] a2) Preparation of the first polypeptide conjugate: The first polypeptide conjugate is obtained by methods such as glycoconjugation, random cysteine ​​conjugation, or bridging, wherein,

[0097] The steps of the sugar coupling preparation method are as follows:

[0098] a2-1) Preparation of the first non-natural glycoengineered polypeptide: The first polypeptide, disaccharide linker and glycoside endonuclease ENGase were mixed, incubated and then purified to obtain the first non-natural glycoengineered polypeptide.

[0099] a2-2) Preparation of the first polypeptide conjugate: Add the non-natural sugar engineered first polypeptide and linker-loader to the reaction system, incubate, and confirm the reaction product by LC-MS to obtain the first polypeptide conjugate;

[0100] The steps of the method for preparing cysteine ​​by random coupling or bridging are as follows:

[0101] a2-1) The first polypeptide and the reducing agent are mixed, incubated, and then the loading reaction is added. The first polypeptide conjugate is then purified.

[0102] b) Mix the first polypeptide conjugate and the second polypeptide in the same reaction system, add a reducing agent, and incubate;

[0103] c) Remove the reducing agent, allow natural oxidation, and purify to obtain a single-loaded heterodimeric polypeptide conjugate;

[0104] d) Preparation of dual-loaded heterodimeric polypeptide conjugates: The dual-loaded heterodimeric polypeptide conjugates are obtained by methods such as sugar conjugation, random cysteine ​​conjugation, or bridging, wherein...

[0105] The steps of the sugar coupling preparation method are as follows:

[0106] d1) Preparation of heterodimeric peptide conjugates containing non-natural sugar engineered second peptides: Mix a single-loaded heterodimeric peptide conjugate with a disaccharide linker and glycoside endonuclease ENGase, incubate and purify to obtain heterodimeric peptide conjugates containing non-natural sugar engineered second peptides.

[0107] d2) Mix the heterodimeric peptide conjugate containing the second polypeptide engineered from non-natural sugars and the second linker-loader in the same reaction system, incubate, and confirm the reaction product by LC-MS to obtain the dual-loaded heterodimeric peptide conjugate.

[0108] The steps of the method for preparing cysteine ​​by random coupling or bridging are as follows:

[0109] d1) Mix the single-loaded heterodimeric peptide conjugate with a reducing agent, incubate, add the loading reaction, and purify to obtain the double-loaded heterodimeric peptide conjugate;

[0110] In step b), the first polypeptide conjugate and the second polypeptide are mixed in a molar ratio of 1:1, the concentration of the reducing agent is in the range of 0.5-100mM, the pH of the reaction system is 6-8, the temperature is 20-40℃, and the time is 0.5-48h.

[0111] In some embodiments, the preparation method includes the following steps:

[0112] a1) Prepare the first polypeptide and the second polypeptide respectively: Construct expression vectors containing the first polypeptide and the second polypeptide respectively, transform the expression vectors into host cells for expression, and purify to obtain the first polypeptide and the second polypeptide, wherein the first Fc region of the first polypeptide and the second Fc region of the second polypeptide contain different amino acid substitutions;

[0113] a2) Preparation of the first polypeptide conjugate and the non-natural glycoengineered second polypeptide: The first polypeptide conjugate is obtained by glycoconjugation, random cysteine ​​conjugation, or bridging methods, wherein...

[0114] The steps of the sugar coupling preparation method are as follows:

[0115] a2-1) Preparation of non-natural glycoengineered first polypeptide and non-natural glycoengineered second polypeptide: Mix the first polypeptide with the disaccharide linker and the glycoside endonuclease ENGase, and mix the second polypeptide with the disaccharide linker and the glycoside endonuclease ENGase. After incubation, purify to obtain the non-natural glycoengineered first polypeptide and the non-natural glycoengineered second polypeptide.

[0116] a2-2) Preparation of the first polypeptide conjugate: Add the non-natural sugar engineered first polypeptide and linker-loader to the reaction system, incubate, and confirm the reaction product by LC-MS to obtain the first polypeptide conjugate;

[0117] The steps of the method for preparing cysteine ​​by random coupling or bridging are as follows:

[0118] a2-1) Preparation of the first polypeptide conjugate: Mix the first polypeptide with a reducing agent, incubate, add a loading reaction, and purify to obtain the first polypeptide conjugate;

[0119] a2-2) Preparation of non-natural glycoengineered second polypeptide: The second polypeptide, disaccharide linker and glycoside endonuclease ENGase were mixed, incubated and then purified to obtain the non-natural glycoengineered second polypeptide;

[0120] b) Mix the first polypeptide conjugate and the non-natural sugar-engineered second polypeptide in the same reaction system, add a reducing agent, and incubate.

[0121] c) Remove the reducing agent, allow natural oxidation, and purify to obtain a single-loaded heterodimeric polypeptide conjugate;

[0122] d) Preparation of dual-loaded heterodimeric peptide conjugates: Mix a single-loaded heterodimeric peptide conjugate and a second linker-loader in the same reaction system, incubate, and confirm the reaction product by LC-MS to obtain dual-loaded heterodimeric peptide conjugates.

[0123] In step b), the first polypeptide conjugate and the non-natural sugar engineered second polypeptide are mixed in a molar ratio of 1:1, the concentration of the reducing agent is in the range of 0.5-100mM, the pH of the reaction system is 6-8, the temperature is 20-40℃, and the time is 0.5-48h.

[0124] In some embodiments, the preparation method includes the following steps:

[0125] a1) Prepare the first polypeptide and the second polypeptide respectively: Construct expression vectors containing the first polypeptide and the second polypeptide respectively, transform the expression vectors into host cells for expression, and purify to obtain the first polypeptide and the second polypeptide, wherein the first Fc region of the first polypeptide and the second Fc region of the second polypeptide contain different amino acid substitutions;

[0126] a2) Preparation of non-natural first polypeptide and non-natural glycoengineered second polypeptide: Mix the first polypeptide with a disaccharide linker and glycoside endonuclease ENGase, and mix the second polypeptide with a disaccharide linker and glycoside endonuclease ENGase. After incubation, purify to obtain non-natural glycoengineered first polypeptide and non-natural glycoengineered second polypeptide, wherein the disaccharide linker structures of the first polypeptide and the second polypeptide are different.

[0127] b) Mix the first non-natural sugar engineered polypeptide and the second non-natural sugar engineered polypeptide in the same reaction system, add a reducing agent, and incubate.

[0128] c-1) Remove the reducing agent, allow natural oxidation, and purify to obtain a heterodimeric polypeptide;

[0129] c-2) Preparation of dual-loaded heterodimeric peptide conjugates: Mix heterodimeric peptides and first linker-loaders and second linker-loaders in the same reaction system, incubate, and confirm the reaction products by LC-MS to obtain dual-loaded heterodimeric peptide conjugates;

[0130] In step b), the first non-natural sugar engineered polypeptide and the second non-natural sugar engineered polypeptide are mixed in a molar ratio of 1:1, the concentration of the reducing agent is in the range of 0.5-100mM, the pH of the reaction system is 6-8, the temperature is 20-40℃, and the time is 0.5-48h.

[0131] In some embodiments, the preparation method includes the following steps:

[0132] a1) Prepare the first polypeptide and the second polypeptide respectively: Construct expression vectors containing the first polypeptide and the second polypeptide respectively, transform the expression vectors into host cells for expression, and purify to obtain the first polypeptide and the second polypeptide, wherein the first Fc region of the first polypeptide and the second Fc region of the second polypeptide contain different amino acid substitutions;

[0133] a2) Preparation of the first non-natural sugar engineered polypeptide: The first polypeptide, disaccharide linker and glycoside endonuclease ENGase were mixed, incubated and then purified to obtain the first non-natural sugar engineered polypeptide.

[0134] b) Mix the first and second non-natural sugar engineered peptides in the same reaction system, add a reducing agent, and incubate.

[0135] c-1) Remove the reducing agent, allow natural oxidation, and purify to obtain a heterodimeric polypeptide;

[0136] c-2) Preparation of single-loaded heterodimeric peptide conjugates: Mix heterodimeric peptides and the first linker-loader in the same reaction system, incubate, and confirm the reaction product by LC-MS to obtain single-loaded heterodimeric peptide conjugates;

[0137] c-3) Preparation of heterodimeric peptide conjugates containing non-natural glycoengineered second peptides: Mix a single-loaded heterodimeric peptide conjugate with a disaccharide linker and glycoside endonuclease ENGase, incubate and then purify to obtain heterodimeric peptide conjugates containing non-natural glycoengineered second peptides.

[0138] d) Preparation of dual-loaded heterodimeric peptide conjugates: Mix heterodimeric peptide conjugates containing a non-natural sugar engineered second peptide and a second linker-loader in the same reaction system, incubate, and confirm the reaction product by LC-MS to obtain dual-loaded heterodimeric peptide conjugates.

[0139] In step b), the first and second non-natural sugar engineering peptides are mixed in a molar ratio of 1:1, the concentration of the reducing agent is in the range of 0.5-100mM, the pH of the reaction system is 6-8, the temperature is 20-40℃, and the time is 0.5-48h.

[0140] In some preferred embodiments, the Fc regions of the first and second polypeptides in the preparation method each contain amino acid substitutions selected from any one of the following groups, wherein the Fc regions numbered EU contain amino acid substitutions selected from any one of the following groups, wherein the EU numbers are: D399R / S364H, K409D / Y349T / K370S; D399K / S364R, K409D / Y349T / K370S; D399K / S364H, K409D / Y349T / K370S; D399K / E356K. K392D / K409D; D399K / E356K, K370D / K409D; S364K / N390P / S400K, S364D / K370D / N390D / K392G / S400D; D399K / E357K, K 392D / K409D; D399K / E357K, K370D / K409D; F405L, K409R; WT, F405L / R409K; T366S / L368A / Y407V / Y349C, T366W / S354C.

[0141] In some preferred embodiments, the preparation method includes the following steps:

[0142] a1) Construct expression vectors containing the first and second polypeptides, respectively. Transform these expression vectors into host cells for expression, and purify to obtain the first and second polypeptides.

[0143] The first polypeptide contains K370S, Y349T, and K409D amino acid substitutions in its first Fc region; the second polypeptide contains S364R and D399K amino acid substitutions in its second Fc region; or...

[0144] The first polypeptide contains T366S, L368A, Y407V, and Y349C amino acid substitutions in its first Fc region; the second polypeptide contains T366W and S354C amino acid substitutions in its second Fc region; or...

[0145] The first polypeptide contains a K409R amino acid substitution in the first Fc region, and the second polypeptide contains an F405L amino acid substitution in the second Fc region.

[0146] The first polypeptide contains K370S, Y349T, and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399R and S364H amino acid substitutions in its second Fc region; or

[0147] The first polypeptide contains K392D and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399K and E356K amino acid substitutions in its second Fc region; or

[0148] The first polypeptide contains K370D and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399K and E356K amino acid substitutions in its second Fc region; or

[0149] The first polypeptide contains S364K, N390P, and S400K amino acid substitutions in its first Fc region, and the second polypeptide contains S364D, K370D, N390D, K392G, and S400D amino acid substitutions in its second Fc region.

[0150] a2) Mix the first polypeptide with the disaccharide linker and the glycoside endonuclease Endo Si or Endo Se2, and mix the second polypeptide with the disaccharide linker and the glycoside endonuclease Endo Si or Endo Se2. After incubation, purify to obtain the non-natural sugar engineered first polypeptide and the non-natural sugar engineered second polypeptide.

[0151] a3) Add a non-natural sugar engineered first polypeptide and a cycloalkyne linker-load to the reaction system, and add a non-natural sugar engineered second polypeptide and a cycloalkyne linker-load to another reaction system. Incubate them separately, and confirm the reaction products by LC-MS to obtain the first polypeptide conjugate and the second polypeptide conjugate, respectively.

[0152] b) Mix the first and second polypeptide conjugates, add 2-mercaptoethylamine hydrochloride (2-MEA), and incubate.

[0153] c) Remove 2-mercaptoethylamine hydrochloride (2-MEA), and oxidize naturally to obtain heterodimeric polypeptide conjugates.

[0154] In some preferred embodiments, the preparation method includes the following steps:

[0155] a1) Construct expression vectors containing the first and second polypeptides, respectively. Transform these expression vectors into host cells for expression, and purify to obtain the first and second polypeptides.

[0156] The first polypeptide contains K370S, Y349T, and K409D amino acid substitutions in its first Fc region; the second polypeptide contains S364R and D399K amino acid substitutions in its second Fc region; or...

[0157] The first polypeptide contains T366S, L368A, Y407V, and Y349C amino acid substitutions in its first Fc region; the second polypeptide contains T366W and S354C amino acid substitutions in its second Fc region; or...

[0158] The first polypeptide contains a K409R amino acid substitution in the first Fc region, and the second polypeptide contains an F405L amino acid substitution in the second Fc region.

[0159] The first polypeptide contains K370S, Y349T, and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399R and S364H amino acid substitutions in its second Fc region; or

[0160] The first polypeptide contains K392D and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399K and E356K amino acid substitutions in its second Fc region; or

[0161] The first polypeptide contains K370D and K409D amino acid substitutions in its first Fc region, and the second polypeptide contains D399K and E356K amino acid substitutions in its second Fc region; or

[0162] The first polypeptide contains S364K, N390P, and S400K amino acid substitutions in its first Fc region, and the second polypeptide contains S364D, K370D, N390D, K392G, and S400D amino acid substitutions in its second Fc region.

[0163] a2) Mix the first polypeptide with the disaccharide linker and the glycoside endonuclease Endo Si or Endo Se2, and mix the second polypeptide with the disaccharide linker and the glycoside endonuclease Endo Si or Endo Se2. After incubation, purify to obtain the non-natural sugar engineered first polypeptide and the non-natural sugar engineered second polypeptide.

[0164] b) Mix the first non-natural sugar engineered peptide and the second non-natural sugar engineered peptide, add 2-mercaptoethylamine hydrochloride (2-MEA), and incubate;

[0165] c-1) Remove 2-mercaptoethylamine hydrochloride (2-MEA), and then naturally oxidize to obtain a non-natural sugar-engineered heterodimeric polypeptide;

[0166] c-2) Add non-natural sugar engineered heterodimeric peptides and linkers-loaders to the reaction system, incubate, and confirm the reaction products by LC-MS to obtain heterodimeric peptide conjugates.

[0167] Thirdly, this disclosure provides heterodimeric polypeptide conjugates prepared by the preparation method of heterodimeric polypeptide conjugates described in the first or second aspect.

[0168] Fourthly, this disclosure provides pharmaceutical compositions comprising the heterodimeric polypeptide conjugate described in the third aspect, and one or more pharmaceutically acceptable carriers.

[0169] Fifthly, this disclosure provides the use of the heterodimeric polypeptide conjugate described in the third aspect or the pharmaceutical composition described in the fourth aspect in the preparation of medicaments for treating and / or preventing diseases. In some embodiments, the disease is a tumor.

[0170] In a sixth aspect, this disclosure provides a method for treating and / or preventing a disease in a subject, comprising administering to the subject the heterodimeric polypeptide conjugate described in the third aspect or the pharmaceutical composition described in the fourth aspect. In some embodiments, the disease is a tumor.

[0171] In a seventh aspect, this disclosure provides the heterodimeric polypeptide conjugates described in the third aspect or the pharmaceutical compositions described in the fourth aspect, for the treatment and / or prevention of diseases, or as medicaments for the treatment and / or prevention of diseases. In some embodiments, the disease is a tumor.

[0172] This disclosure provides a versatile, feasible, and tunable drug loading (DAR value) homogeneous heteropeptide conjugate preparation scheme. By preparing the peptide-coupled load and combining it with in vitro antibody recombination technology, three forms of heteropeptide conjugates can be prepared, including: single-target dual-load, dual-target single-load, and dual-target dual-load heterodimeric peptide conjugates; especially for dual-target dual-load peptide conjugates, it overcomes the technical barriers in the preparation of existing technologies. Furthermore, in the preparation of heterodimeric peptide conjugates, the order of the peptide-load coupling steps and the in vitro recombination steps can be adjusted: either two peptide conjugates can be prepared separately and then recombinated; or one peptide conjugate can be prepared first, recombined with another peptide, and then coupled with another load; or the peptide can be recombined first, and then the load can be coupled. This disclosure combines the advantages of antibody engineering and payload conjugation technologies, enabling individual adjustment of the DAR value of any payload; furthermore, the asymmetric structure makes it possible to prepare dual-load ADCs (such as DAR1+1 ADCs) with ultra-low DAR values. When using site-directed conjugation methods (such as glycosyl-based site-directed conjugation methods), this disclosure can prepare highly uniform target components and is suitable for a wide range of applications. In addition, this disclosure can also obtain dual-target dual-load peptide conjugates with better uniformity and no mismatch between light and heavy chains by employing techniques to avoid Fab mismatch. Attached Figure Description

[0173] Figure 1: Preparation method of heterodimeric polypeptide conjugates. Methods for preparing heterodimeric polypeptide conjugates with the same or different loadings, respectively: first polypeptide conjugate × second polypeptide conjugate, first polypeptide conjugate × second polypeptide, first polypeptide conjugate × second polypeptide, first polypeptide conjugate × second polypeptide, and first polypeptide conjugate × second polypeptide. This indicates the location of the load coupling in the modified first or second polypeptide. ☆ indicates Fc region modification regions, which make the heterogeneous interactions of Fc stronger than the homogeneous interactions.

[0174] Figure 2: Method steps for preparing heterodimeric polypeptide conjugates by combining the first polypeptide conjugate with the second polypeptide conjugate, using different conjugation methods. For the same or different loads, they represent site-to-site coupling, mercapto coupling, mercapto coupling, and mercapto coupling, respectively. ☆ indicates Fc region modification regions, which make the heterogeneous interactions of Fc stronger than the homogeneous interactions.

[0175] Figure 3: Method steps for preparing heterodimeric polypeptide conjugates by first polypeptide conjugate × second polypeptide or first polypeptide conjugate × second polypeptide modified by conjugation. For the same or different loads. This indicates the location of the load-bearing coupling in the second polypeptide after coupling modification. ☆ indicates Fc region modification regions, which make the heterogeneous interactions of Fc stronger than the homogeneous interactions.

[0176] Figure 4: Method steps for preparing heterodimeric polypeptide conjugates by coupling modified first polypeptide × second polypeptide or coupling modified first polypeptide × coupling modified second polypeptide. For the same or different loads. This indicates the location of the load-bearing coupling in the first or second polypeptide after coupling modification. ☆ indicates Fc region modification regions, which make the heterogeneous interactions of Fc stronger than the homogeneous interactions.

[0177] Figure 5: Mass spectrometry detection of the preparation process of dual-load ADC dpADC3. Uniform dpADC3 was produced after in vitro recombination of Ab7-G35-LP1 and Ab8-G35-LP2.

[0178] Figure 6: Efficacy of dual-load ADC dpADC3 in the NCI-N87 xenograft model. Detailed Implementation

[0179] 1. Terminology

[0180] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated to be incorporated by reference.

[0181] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0182] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0183] The term "peptide conjugate" refers to any conjugate formed by covalently linking a peptide containing an Fc region to a payload. The peptide may contain a portion that binds to a specific target. This target-binding portion can be an antibody or its antigen-binding fragment, such as a monoclonal antibody, bispecific antibody, or polyclonal antibody; it can be the extracellular region of a ligand-binding cell receptor protein or a mutant thereof. The payload can be a cytotoxin, small molecule drug, near-infrared or fluorescent probe, peptide, RNA and related drugs, radiolabeled isotopes, contrast agents, and magnetic resonance imaging agents, etc. The resulting conjugate can be used for treatment or detection.

[0184] The term "heterodimeric peptide conjugate," also known as "heteropeptide conjugate" or "heterodimeric peptide conjugate," refers to a conjugate composed of a heterodimeric peptide containing a heterodimeric Fc region and a loading vector, wherein the heterodimeric peptide contains a first peptide containing a first Fc region and a second peptide containing a second Fc region, and the first peptide and the second peptide form a heterodimeric peptide through the first Fc region and the second Fc region. The first peptide is coupled to the loading vector to form a first peptide conjugate, and the second peptide is coupled to the loading vector to form a second peptide conjugate. Heterodimeric peptide conjugates can be prepared from the first peptide conjugate and the second peptide conjugate, or from a coupled-modified second peptide or second peptide; or, from a coupled-modified first peptide and a coupled-modified second peptide or second peptide prepared into a coupled-modified heterodimeric peptide, which is then coupled to the loading vector. Heterodimeric peptide conjugates can be peptide conjugates in which both a first peptide and a second peptide are coupled, peptide conjugates in which the first peptide is coupled but the second peptide is not, or peptide conjugates in which the first peptide is not coupled but the second peptide is. The coupled heterodimeric peptide can be prepared from a coupled first peptide and a coupled second peptide, or vice versa. The coupled heterodimeric peptide can be a peptide in which both the first and second peptides are modified, or a peptide in which the first peptide is modified but the second peptide is not.

[0185] The terms "dual-target dual-load heterodimeric peptide conjugate" or "dual-target dual-load heterodimeric peptide conjugate" refer to heterodimeric peptide conjugates in which the first and second peptides contain portions that bind to different targets and are coupled with different loads. The terms "single-target dual-load heterodimeric peptide conjugate" or "single-target dual-load heterodimeric peptide conjugate" refer to heterodimeric peptide conjugates in which the first and second peptides contain portions that bind to the same target and are coupled with different loads. The terms "dual-target single-load heterodimeric peptide conjugate" or "dual-target single-load heterodimeric peptide conjugate" refer to heterodimeric peptide conjugates in which the first and second peptides contain portions that bind to different targets and are coupled with the same load. The first and second peptides can be coupled with the same or different loads through the same or different coupling methods and linkers.

[0186] The term "coupled-modified polypeptide" refers to a polypeptide containing an Fc region that can be coupled through methods including, but not limited to, modification of one or more natural and / or non-natural amino acids (i.e., amino acid substitution, deletion, or insertion), chemical modification, enzymatic modification, or a combination thereof. This coupling modification creates new loading coupling sites, alters existing coupling sites, and / or removes existing coupling sites, resulting in a coupled-modified polypeptide (e.g., removing existing glycosylation sites, adding cysteine ​​coupling sites, etc.). The coupling modification allows the polypeptide to have different coupling sites. A first polypeptide containing a first Fc region is coupled to form a coupled-modified first polypeptide, and a second polypeptide containing a second Fc region is coupled to form a coupled-modified second polypeptide. A coupled-modified first polypeptide coupled with a loading device forms a first polypeptide conjugate, and a coupled-modified second polypeptide coupled with a loading device forms a second polypeptide conjugate.

[0187] The first polypeptide and / or the second polypeptide can be a protein comprising a target-binding region, the target-binding region including an Fc region, and a target-binding domain fused to the Fc region (including, but not limited to,, for example, the heavy and / or light chain variable regions of an antibody that can bind to the target, an antigen-binding fragment of an antibody; or, a soluble portion of a ligand or receptor that can bind to the target). The target-binding domains contained in the first polypeptide and the second polypeptide may target the same or different targets. The first polypeptide and / or the second polypeptide may also be a protein comprising an Fc region.

[0188] In some embodiments, both the first and second peptides are antibodies, preferably full-length antibodies, and the resulting heterodimeric peptide is a bispecific antibody containing both a first and a second Fc region. In some embodiments, only the first or second peptide is a full-length antibody, and the other peptide is not a full-length antibody, such as lacking the variable Fc region, along with other target-binding domains, resulting in a heterodimeric peptide that is a peptide combining an antibody containing the first Fc region and a fusion protein containing the second Fc region. In some embodiments, neither the first nor the second peptide is a full-length antibody, and the resulting heterodimeric peptide is a fusion protein containing both the first and second Fc regions.

[0189] The payload can be coupled to a first polypeptide and a second polypeptide, or to either the first polypeptide or the second polypeptide, to form a first polypeptide conjugate and / or a second polypeptide conjugate. The type and / or number of payloads coupled to the first and second polypeptides can be the same, partially the same, or different. In some embodiments, the first and second polypeptides target different molecules, and the number and / or type of payloads coupled to each polypeptide chain are the same. In some embodiments, the first and second polypeptides target the same molecules, and the number and / or type of payloads coupled to each polypeptide chain are different. In some embodiments, the first and second polypeptides target different molecules, and the number and / or type of payloads coupled to each polypeptide chain are also different.

[0190] The term "target-binding region" refers to any portion of a heterodimeric polypeptide conjugate that binds to a specific target (e.g., an antigen). The target-binding region can contain, for example, an antibody or immunoglobulin itself or its antigen-binding fragment. It can also be a soluble portion containing, for example, a ligand or receptor that binds to the target. The "target-binding region" includes the Fc region. The first and second target-binding regions can have the same or different structures. When the first and second target-binding regions have the same structure, for example, both containing Fab fragments linked to the Fc region, it is called "symmetric first and second target-binding regions." When the first and second target-binding regions have different structures, for example, the first target-binding region contains a Fab fragment linked to the Fc region, and the second target-binding region contains a VHH, scFv, or other antigen-binding fragment linked to the Fc region, or contains other proteins linked to the Fc region, such as ligands or receptors, it is called "asymmetric first and second target-binding regions."

[0191] The term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, specifically the two polypeptide chains that form a dimer containing the self-binding C-terminal constant region of the immunoglobulin heavy chain. While the boundaries of the Fc region of the IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is typically defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain; for example, the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0192] The Fc region includes the native Fc region and variant Fc regions. The Fc region can be derived from any IgG, preferably from mammalian or human IgG, such as IgG1, IgG2, IgG3, or IgG4 Fc regions. The amino acid sequence of the human IgG1 Fc region begins at the hinge region and terminates at the carboxyl terminus of the CH3 region. The native or wild-type human IgG1 Fc region is intended to encompass these native allelic forms herein. In this document, the Fc region can be the isolated region or the region within an antibody, antibody fragment, or Fc fusion protein. The terms “Fc region protein” or “Fc region polypeptide” are used interchangeably herein to refer to a protein or polypeptide containing the Fc region. The terms “Fc region variant” and “variant Fc region” are used interchangeably to refer to an Fc region in which one or more amino acid modifications (i.e., amino acid substitutions, deletions, and / or insertions) are introduced at any position within the Fc region relative to the unmodified Fc region (i.e., the parental Fc region).

[0193] The term "CH3 domain" or "CH3 region" refers to the third constant region of the heavy chain. The CH3 domain can originate from the heavy chains of human IgG1, IgG2, IgG3, or IgG4.

[0194] The terms "first Fc region" and "second Fc region" refer to two distinct Fc regions in a heterodimeric polypeptide conjugate that can pair with each other. The heterodimeric interaction between the first and second Fc regions is stronger than the homodimeric interaction between each Fc region individually. When used herein, the term "heterodimeric interaction between the first and second Fc regions" refers to the interaction between the first and second Fc regions in a first Fc / second Fc region heterodimeric protein. When used herein, the term "homogeneous dimeric interaction of the first Fc region" refers to the interaction between the first Fc region and another first Fc region in a first Fc / first Fc region homodimeric protein; "homogeneous dimeric interaction of the second Fc region" refers to the interaction between the second Fc region and another second Fc region in a second Fc / second Fc region homodimeric protein. The terms "same origin" and "different origin" used in this article are a set of relative concepts. They can refer to different elements in a construct having the same or different origins, or they can refer to multiple elements that originally had the same origin ("same origin") becoming "different origins" after the construct is completed. Some of these elements have been modified and changed compared to the other original elements that have not been modified.

[0195] The first Fc region and the second Fc region have different sequences, meaning that the first Fc region and / or the second Fc region contain one or more amino acid modifications (i.e., amino acid substitution, deletion, or insertion) in the wild-type Fc region; wherein, one or more amino acid substitutions include amino acid changes that alter the heterodimerization properties of the Fc region, such that the heterodimer interaction between the first Fc region and the second Fc region is stronger than the homodimer interaction between the first Fc region and the second Fc region, including but not limited to one or more amino acid substitutions based on charge interaction, one or more amino acid substitutions based on non-electrostatic interaction, and / or one or more amino acid substitutions based on other interactions.

[0196] The term "charge-based interaction" refers to interactions based on positive and negative charges, or interactions based on positive and uncharged charges, or interactions based on negative and uncharged charges. Families of amino acid residues with similar side chains have been defined in the art, including amino acids with basic, positively charged side chains (e.g., lysine, arginine, histidine), amino acids with acidic, negatively charged side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid substitutions can also be introduced into the first and second Fc regions based on other non-electrostatic interactions, such as spatial structure, hydrophobicity, hydrogen bonding, and aromatic stacking, to enhance heterodimeric pairing between the first and second Fc regions. In some embodiments, amino acid substitutions can be introduced by replacing native amino acid residues in the CH3 domains of the first and second Fc regions, respectively. In some embodiments, one or more amino acid substitutions can be introduced into the first and second Fc regions.

[0197] The term "amino acid substitution" or "replacement" refers to replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, S32A means that serine at position 32 is replaced by alanine; K409A / G / R / H means that lysine at position 409 is replaced by alanine, glycine, arginine, or histidine; WT means that the wild type has not undergone substitution.

[0198] Techniques for amino acid substitution that enable a stronger heterodimeric interaction between the first Fc region and the second Fc region than the homodimeric interaction between the first and second Fc regions, including but not limited to various Fc heterodimer techniques, such as Knobs-into-Holes (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329), and charge matching (Amgen, WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2), DuoBody, etc. TM (Genmab A / S, WO2011131746), LUZ-Y, SEEDbody, Biclonic, bispecific IgG1 and IgG2, Azymetric scaffold and differential protein A affinity, etc. (see, e.g., Ulrich Brinkmann et al., 2016, mAbs 9:2, 182-212, doi.org / 10.1080 / 19420862.2016.1268307; Xu Han et al., 2024, Immunology 173:3, 562-574, doi:10.1111 / imm.13845, and the references cited therein).

[0199] In some implementations, Fc heterodimer technology is DuoBody TM Platform. Amino acid substitutions in the first Fc region and / or the second Fc region may be located at residues 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy chain constant region (EU number, see WO2011131746). Furthermore, amino acid substitutions in the first and second Fc regions may be located at different residues. For example, the first Fc region may contain the mutation F405L (EU number), or one of the mutations F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y, while the second Fc region may contain the mutation K409R (EU number).

[0200] In some embodiments, the Fc heterodimer technology is the Knobs-into-Holes technology. At the sites where the two heavy chains directly interact within the CH3 domains of the first and second Fc regions, an amino acid with a small side chain (Hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (Knob) is introduced into the corresponding interacting residue site on the other heavy chain. For example, the "Knob chain" contains a T366W (EU number) substitution and the "Holes chain" contains T366S, L368A, and Y407V (EU number) substitutions. In some embodiments, additional interchain disulfide bridges are introduced between the CH3 domains, for example, by introducing a Y349C (EU number) substitution into the "Knob chain" and an E356C (EU number) or S354C (EU number) substitution into the "Holes chain." In some embodiments, an R409D or K370E (EU number) substitution is introduced into the "Knob chain," and a D399K or E357K (EU number) substitution is introduced into the "Holes chain." In some embodiments, Y349C and T366W (EU designations) are introduced to replace the first chain, and E356C, T366S, L368A, and Y407V (EU designations) are introduced to replace the corresponding chains. In some embodiments, Y349C and T366W (EU designations) are introduced to replace the first chain, and S354C, T366S, L368A, and Y407V (EU designations) are introduced to replace the corresponding chains.

[0201] In some implementations, the first Fc region and the second Fc region contain the following mutations (EU numbers): T366S / L368A / Y407V, T366W; S354C / T366W, Y349C / T366S / L368A / Y407V; T366Y, Y407T; T366W, Y407A; T394W, F405A; T366Y / F405A, T394W / Y407T; T366W / F405W, T394S / Y407A; F405W, T394S; D399C, K392C; T366W / D399C, T366S / L368A / K392C / Y407V; T366W / K39 2C, T366S / L368A / D399C / Y407V; S354C / T366W, Y349C / T366S / L368A / Y407V ; Y349C / T366W, S354C / T366S / L368A / Y407V; E356C / T366W, Y349C / T366S / L 368A / Y407V; Y349C / T366W, E356C / T366S / L368A / Y407V; E357C / T366W, Y34 9C / T366S / L368A / Y407V; Y349C / T366W, E357C / T366S / L368A / Y407V; T366K , L351D; T366K, Y349E / D; T366K, L368E / D; T366K, L351D / Y349D / R355D; T36 6K, Y349E / D / L368E / D / L351D; T366K / L351K, L351D; T366K / L351K, Y349E / D ;T366K / L351K, L368E / D; T366K / L351K, Y349E / D / L368E / D / L351D; T366K / L 351K, L351D / Y349D / R355D; L351K, L351D; K370E / D399K / K439D, D356K / E35 7K / K409D; K409D, D399K; K409E, D399K; K409E, D399R; K409D, D399R; D339K , E356K; E356K / D399K, K392D / K409D; E356K, K439E; E356D, K439R; E356K / D 399K, K409D / K439D; E357K, K370D; E357K / D399K, K370D / K409D; E356K / E35 7K / D399K, K370D / K392D / K409D; E356K / E357K / D399K, K370E / K392D / K409D;E357K / D399K,K392D / K409D;D399K / R,K392D;K392D / K409D,D399K;K360D / K409D,D399K;S364R / D399K,K370S / Y349T / K409D;S364R / D399K / E357N / T350V,K370S / Y349T / Q347D / K409D / T350V;L351Y / S400E / F405A / Y407V / T350V,T366L / N390R / K392M / T394W / T350V;K409E / T411R,D399K / K370T;S364R / D399K / E357N,K370S / Y349T / Q347D / K409D;S364R / D399K,K370S / Y349T / Q347D / K409D;F405L,K409R;WT,F405L / R409K;T350I / K370T / F405L,K409R;K370W,K409R;D399A / F / G / H / I / L / M / N / R / S / T / V / W / Y,K409R;T366A / D / E / F / G / H / I / L / M / Q / V / Y,K409R;L368A / D / E / G / H / N / R / S / T / V / Q,K409A / G / R / H;D399F / H / K / R / Q,K409A / G / R / H;F405I / K / L / S / T / V / W,K409A / G / R / H;Y407L / W / Q,K409A / G / R / H;D360K,K402D;D403K,K419D;E357K,K370E;K249E / K288E,E236K / D278K;H435R,WT;S364H,Y349T;S364H / F405A,Y349T / T394F;S364H,Y349T;D399R / S364H,K409D / Y349T / K370S;D399K / S364H,K409D / Y349T / K370S;D399K / E356K,K370D / K409D;S364K / N390P / S400K,S364D / K370D / N390D / K392G / S400D。;

[0202] In some preferred embodiments, the first Fc region and the second Fc region contain the following mutations (EU numbers): D399R / S364H, K409D / Y349T / K370S; D399K / S364R, K409D / Y349T / K370S; D399K / S364H, K409D / Y349T / K370S; D399K / E356K, K392D / K409D; D399K / E356K, K37 0D / K409D; S364K / N390P / S400K, S364D / K370D / N390D / K392G / S400D; D399K / E357K, K392D / K409D; D39 9K / E357K, K370D / K409D; F405L, K409R; WT, F405L / R409K; T366S / L368A / Y407V / Y349C, T366W / S354C.

[0203] In some implementations, other techniques for modifying CH3 in the Fc region to enhance heterodimerization are considered as alternatives and described. For example, there are several ways to modify the CH3 domain of the Fc region to enhance heterodimerization, which are detailed in, for example, WO2006106905, WO2020216878, WO2022150785, WO2022143912, WO2023049732, etc.

[0204] In this disclosure, the first Fc region and the second Fc region refer to general markers that represent two heterologous Fc region sequences, and do not represent the order in the heterodimeric polypeptide. They should not be construed as marking a specific or particular part of the heterodimeric polypeptide conjugate provided in this disclosure. The substituted amino acids in the first Fc region and the second Fc region can be reversed, that is, any substituted amino acid in the first Fc region can alternatively be in the second Fc region, and any substituted amino acid in the second Fc region can alternatively be in the first Fc region.

[0205] The term "target-binding domain" refers to any domain that can bind to a target, such as the variable regions of the heavy and / or light chains of an antibody that binds to the target, an antigen / target binding fragment, or a soluble portion of a ligand or receptor that can bind to the target. The target can be any molecule located on tumor cells or effector cells. Targets can be, for example, the following antigens or any combination thereof: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Big3, SPARC, BCR, CD79, CD37, EGFrRvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, MRP3, TROP2, Nectin-4, CLDN 18.2, CLDN6, CDH6, B7-H3, ROR1, PSMA, PD-L1, FRα, DLL3, CDH17, 5T4, and tissue. Factor, PTK7, BCMA, NaPi2b, B7-H4, LIV-1, CEACAM5, CD22, CD33, MSLN, CD30, GPC3, IL3RA, FGFR2b, CDCP1, CD73, uPARAP, KIT, IL2RA, ICAM-1, GUCY2C, CDH3, CD79b, CD70, VEGFR2, VEGF, TFR1, S TEAP1, SEZ6, NCAM1, MUC16, LRRC15, IL13RA2, DLK1, CLL-1, CD45, CD37, BCL2L1, KIF11, ITGB6, IL1RAP, FLT3, FGFR2, EphA2, DPP-4, CD7, CD47, TRAILR2, TLR7, Tau, Tumor-associated carbohydrate antigen (Tumor-associated carbohydrate (antigen), SORT1, SLC1A5, SDC1, PD-1, NMT, NA+ / K+ATPASE, LY6E, IGF-1R, HSP70, glucocorticoid receptor (GR), FGFR3, ENPP3, CD105, CTLA4, CLDN1, CEACAM6, CD228, CD147, ADAM9, AMHR2, ALPP, ALPG or LY6G6D, etc.; and / or, CD3, CD4, CD8, CD40, CD25, CD28, CD16, CD89, CD32, CD64, FcεRI or CD1, etc.

[0206] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, human antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, etc., as long as they exhibit the desired biological activity. The term "immunoglobulin" is used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. The term "antibody fragment" contains at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its epitope, or a fusion protein product further derived from such fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies (nanobodies, VHH), linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments.

[0207] In some embodiments, the first polypeptide and / or the second polypeptide is an antibody (i.e., a first antibody and / or a second antibody) that further comprises one or more or all of the antibody's other regions, namely a CH1 region, a hinge region, a VH region, a CL region, and / or a VL region. In some embodiments, the first polypeptide and / or the second polypeptide comprises a first heavy chain H1 and a first light chain L1, a second heavy chain H2, and a second light chain L2; wherein H1 and H2 contain a CH1 domain, and L1 and L2 contain a CL domain. In some embodiments, the first polypeptide is a full-length antibody. In some embodiments, the second polypeptide is a full-length antibody. In some embodiments, both the first polypeptide and the second polypeptide are antibodies, preferably full-length antibodies, and bind to different epitopes. In such embodiments, the resulting heterodimeric polypeptide is a bispecific antibody, and the epitopes may be located on different antigens or the same antigen. In such implementations, techniques to avoid Fab mismatch are employed to improve the correct pairing of heavy and light chains in bispecific antibodies, including but not limited to CrossMab (Roche, WO2011117329), common light chain (Crucell / Merus, US7262028), or common heavy chain (K Bodies by Novlmmune, WO2012023053) and DuetMab (Medimmune, US9527927), etc. (see, for example, Ulrich Brinkmann et al., 2016, mAbs 9:2, 182-212, doi.org / 10.1080 / 19420862.2016.1268307, and the references cited therein).

[0208] In some implementations, DuetMab technology is used to improve the correct pairing of heavy and light chains in bispecific antibodies by employing techniques to avoid Fab mismatch. DuetMab technology increases the effectiveness of homologous heavy and light chain pairing by replacing the native disulfide bonds in one of the CH1-CL interfaces with engineered disulfide bonds. Disclosures related to DuetMab can be found, for example, in U.S. Patent No. 9,527,927, which is incorporated herein by reference in its entirety. In some embodiments, the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 comprise: (a) a modified CH1 domain, wherein the modified CH1 domain comprises a substitution of a naturally occurring non-cysteine ​​amino acid for cysteine; and (b) a modified corresponding CL domain, wherein the modified CL domain comprises a substitution of a naturally occurring non-cysteine ​​amino acid for cysteine, wherein: (i) the first heavy chain H1 comprises the modified CH1 domain and the first light chain L1 comprises the modified corresponding CL domain; and / or (ii) the second heavy chain H2 comprises the modified CH1 domain and the second light chain L2 comprises the modified corresponding CL domain. In some embodiments, the substituted cysteine ​​in the modified CH domain resulting from the substitution of a naturally occurring non-cysteine ​​amino acid for cysteine ​​and the substituted cysteine ​​in the modified corresponding CL domain resulting from the substitution of a naturally occurring non-cysteine ​​amino acid for cysteine ​​can form a disulfide bond.

[0209] In some implementations, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following mutations (EU numbers):

[0210] (Ai)IgG1: a. CH1 domain, in which the 141st amino acid is replaced with cysteine ​​and the 220th cysteine ​​is replaced with a non-cysteine ​​amino acid; b. CL domain, in which the 116th amino acid is replaced with cysteine ​​and the 214th cysteine ​​is replaced with a non-cysteine ​​amino acid.

[0211] (A-ii)IgG2: a. CH1 domain, wherein the amino acid at position 141 is replaced with cysteine, and the cysteines at positions 131, 219 and / or 220 are replaced with non-cysteine ​​amino acids; b. CL domain, wherein the amino acid at position 116 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid;

[0212] (A-iii)IgG2: a. CH1 domain, wherein the amino acid at position 141 is replaced with cysteine, and the cysteines at positions 131 and 220 are replaced with non-cysteine ​​amino acids; b. CL domain, wherein the amino acid at position 116 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with non-cysteine ​​amino acids.

[0213] (A-iv)IgG3: a. CH1 domain, in which the 141st amino acid is replaced by cysteine ​​and the 131st cysteine ​​is replaced by a non-cysteine ​​amino acid; b. CL domain, in which the 116th amino acid is replaced by cysteine ​​and the 214th cysteine ​​is replaced by a non-cysteine ​​amino acid.

[0214] (Av)IgG4: a. CH1 domain, in which the 141st amino acid is replaced by cysteine ​​and the 131st cysteine ​​is replaced by a non-cysteine ​​amino acid; b. CL domain, in which the 116th amino acid is replaced by cysteine ​​and the 214th cysteine ​​is replaced by a non-cysteine ​​amino acid.

[0215] (Bi)IgG1: a. CH1 domain, wherein the amino acid at position 168 is replaced with cysteine ​​and the cysteine ​​at position 220 is replaced with a non-cysteine ​​amino acid; b. CL domain, wherein the amino acid at position 164 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0216] (B-ii)IgG2: a. CH1 domain, wherein the amino acid at position 168 is replaced with cysteine, and the cysteines at positions 131, 219 and / or 220 are replaced with non-cysteine ​​amino acids; b. CL domain, wherein the amino acid at position 164 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid;

[0217] (B-iii)IgG2: a. CH1 domain, wherein the amino acid at position 168 is replaced with cysteine, and the cysteines at positions 131 and 220 are replaced with non-cysteine ​​amino acids; b. CL domain, wherein the amino acid at position 164 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with non-cysteine ​​amino acids.

[0218] (B-iv)IgG3: a. CH1 domain, in which the amino acid at position 168 is replaced with cysteine ​​and the cysteine ​​at position 131 is replaced with a non-cysteine ​​amino acid; b. CL domain, in which the amino acid at position 164 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0219] (Bv)IgG4: a. CH1 domain, in which the amino acid at position 168 is replaced with cysteine ​​and the cysteine ​​at position 131 is replaced with a non-cysteine ​​amino acid; b. CL domain, in which the amino acid at position 164 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0220] (Ci)IgG1: a. CH1 domain, in which the amino acid at position 126 is replaced with cysteine ​​and the cysteine ​​at position 220 is replaced with a non-cysteine ​​amino acid; b. CL domain, in which the amino acid at position 121 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0221] (C-ii)IgG2 contains a. a CH1 domain, wherein the amino acid at position 126 is replaced with cysteine, and the cysteines at positions 131, 219, and 220 are replaced with non-cysteine ​​amino acids; b. a CL domain, wherein the amino acid at position 121 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid;

[0222] (C-iii)IgG2 contains a. a CH1 domain, wherein the amino acid at position 126 is replaced with cysteine, and the cysteines at positions 131 and 220 are replaced with non-cysteine ​​amino acids; b. a CL domain, wherein the amino acid at position 121 is replaced with cysteine, and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0223] (C-iv)IgG3 contains a. CH1 domain, in which the amino acid at position 126 is replaced with cysteine ​​and the cysteine ​​at position 131 is replaced with a non-cysteine ​​amino acid; b. CL domain, in which the amino acid at position 121 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0224] (Cv)IgG4 contains a. a CH1 domain, wherein the amino acid at position 126 is replaced with cysteine ​​and the cysteine ​​at position 131 is replaced with a non-cysteine ​​amino acid; b. a CL domain, wherein the amino acid at position 121 is replaced with cysteine ​​and the cysteine ​​at position 214 is replaced with a non-cysteine ​​amino acid.

[0225] In some preferred embodiments, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following mutations (EU numbers): CH1: F126C / C220V, CL: S121C / C214V.

[0226] In some embodiments, the first polypeptide and / or the second polypeptide further contain a combination of mutations that facilitate the separation of polypeptide heterodimers, including but not limited to various techniques for separating and purifying heterodimers, such as RF mutation techniques (H435R, Y436F, see WO2016018740A2, etc.), TLQ mutation techniques (T307P, L309Q, Q311R, see WO2018224951A2, etc.), M428G / N434A mutation techniques (see WO2014049003A1, etc.) or kappa-lambda body techniques (see WO2012023053A2, etc.) (see, for example, Ulrich Brinkmann et al., 2016, mAbs 9:2, 182-212, doi.org / 10.1080 / 19420862.2016.1268307, and the references cited therein).

[0227] The term "fusion protein" refers to polypeptide domains, each with a specific function, linked together directly or tandemly via one or more linkers by peptide bonds. As used herein, "linker" refers to any tool used to connect two polypeptide domains. Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. Polypeptide linkers are preferably non-immunogenic and flexible, such as those containing serine and glycine sequences. Depending on the specific construct, the linker can be long or short. In some embodiments, the first polypeptide and / or the second polypeptide is a fusion protein, which also contains a soluble portion of a ligand or receptor that binds to the target. In some embodiments, the first polypeptide is a fusion protein. In other embodiments, the second polypeptide is a fusion protein. In an important embodiment, the first polypeptide is an antibody, the second polypeptide is a fusion protein, and they bind to different targets. In such embodiments, the resulting heterodimeric polypeptide is a fusion protein, and the target can be a different antigen or a different epitope of the same antigen.

[0228] In this disclosure, the first polypeptide and the second polypeptide, the first antibody and the second antibody, the first heavy chain / first light chain and the second heavy chain / second light chain, the antigen-binding fragment of the first antibody and the antigen-binding fragment of the second antibody, the first fusion protein and the second fusion protein are general labels that represent two polypeptide sequences or domains contained in a heterodimeric polypeptide conjugate, and do not represent their order in the heterodimeric polypeptide conjugate. They should not be construed as labeling a specific or particular part of the heterodimeric polypeptide conjugate provided in this disclosure.

[0229] The term "payload," also known as "warhead drug molecule" or "payload drug," refers to a substance with preventative or therapeutic effects on diseases. In peptide drug conjugates, the drug typically refers to a cytotoxic drug, a chemical molecule that strongly disrupts the normal growth of tumor cells. The payload can be a cytotoxin, small molecule drug, near-infrared or fluorescent probe, peptide, RNA and related drugs, radiolabeled isotopes, contrast agents, and MRI agents, etc. In some embodiments, the small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, DXd, eczema, docamycin, muscarinic acid, PBDs, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of the above compounds, or the small molecule drug may be a radiotherapy agent.

[0230] The term "linker" refers to a chemical structural fragment or bond that is linked to a polypeptide at one end and to a drug at the other end. Linkers can also be linked to other linkers before being attached to a drug. Linkers can be cleavable linkers, non-cleavable linkers, or combinations thereof.

[0231] The term "drug-linker conjugate" is also called load-linker conjugate or linker-load conjugate, and has the same meaning in this disclosure.

[0232] The term "coupling" broadly refers to a reaction in which two molecules combine by forming a new chemical bond; in this article, it specifically refers to the process of transferring a loading onto a polypeptide to form a polypeptide conjugate. The coupling mode and linker of the first polypeptide coupling loading can be the same, partially the same, or different. The coupling mode and linker of the second polypeptide coupling loading can be the same, partially the same, or different. The coupling mode and linker of the coupling loading between the first and second polypeptides can be the same, partially the same, or different. The coupling mode can be any coupling mode well known in the art, including but not limited to those described in Stephen J. Walsh et al., 2021, Chem Soc Rev 50:2, 1305-1353, doi:10.1039 / d0cs00310g.

[0233] The term "random conjugation method" refers to a method of drug conjugation at some or all of several sites on a peptide, typically based on the conjugation of naturally occurring, unmodified amino acid active residues of the peptide. In antibody-drug conjugates, random conjugation methods usually refer to methods that utilize the amino residues of lysine or the thiol residues of cysteine ​​for loading conjugation.

[0234] The term "bridging" refers to a method of drug coupling by covalently linking two thiol groups using a coupling agent. The coupling agent can bridge the two thiol reactive groups or it can bridge two thiol groups simultaneously using a single linker. Any of the functional groups reacting with the thiol groups can be independently selected from maleimide, 2-methylsulfonyl pyrimidine, halogen, halogen-substituted functional groups, alkanes, ketones, sulfonyl alkenes, silanes, isocyanates, or norbornene. Exemplary bridging methods and linkers include, for example, those described in CN107921030B and CN110088086B, which are incorporated herein by reference in their entirety and for all purposes.

[0235] The term "site-directed conjugation" refers to a method that enables drug conjugation at specific sites on a peptide. These specific sites can be conserved amino acids or residues of the peptide, sites introduced through mutation or modification, or conserved post-translational modification sites. In antibody-drug conjugates, site-directed conjugation typically refers to methods that, by controlling the introduction of specific sites, achieve a highly homogeneous drug-antibody ratio compared to random conjugation methods.

[0236] The term "glycosyl-based conjugation" refers to methods that modify the glycosyl groups of a peptide to achieve payload conjugation. In antibody-drug conjugates, glycosyl-based conjugation typically refers to modifying the conserved N-glycosylation at the N297 position to achieve payload conjugation. This modification can be performed enzymatically or chemically.

[0237] The term "enzyme" refers to a biological catalyst composed of polypeptides, ribonucleic acid, or complexes thereof, which can catalyze specific chemical reactions. In this article, it specifically refers to polypeptides or proteins that can catalyze specific modifications to polypeptides and their post-translational modification sites.

[0238] The term "modified enzyme containing a catalytic domain" refers to a modified enzyme that contains the same or similar catalytic domain as the target enzyme, thereby enabling it to perform the same or similar functions.

[0239] The term "engineered cysteine-based coupling method" refers to a method that uses genetic engineering techniques to insert or mutate cysteine ​​residues into a polypeptide, and then uses these cysteine ​​residues to achieve payload coupling. For example, it could be based on THIOMAB. TM Technology. A “cysteine-modified antibody” or “cysteine-modified antibody variant” (ThioMab) refers to an antibody in which multiple residues are replaced by cysteine ​​residues. The thiol group of a cysteine-modified antibody can be conjugated to a drug module (e.g., via a linker) to form a ThioMab antibody-drug conjugate (TDC). In some embodiments, the substituted residues are present at accessible sites on the antibody. For example, ThioMab antibodies may contain light chain components such as G64C, I106C, R108C, K149C, or R142C (Kabat designation) or heavy chain components such as HC-D101C, HC-V184C, or HC-T205C (Kabat designation); or HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, or HC-Q438C (EU designation; HC-A136C according to the Kabat designation is HC-A140C according to the EU designation). In some embodiments, the ThioMab antibody has a single cysteine ​​mutation in either the heavy or light chain, such that each full-length antibody (i.e., an antibody having two heavy chains and two light chains) has two modified cysteine ​​residues. Exemplary methods for preparing cysteine-modified antibodies include, but are not limited to, the methods described, for example, in WO2009052249A1 and WO2016040856A1, which are incorporated herein by reference in their entirety and for all purposes.

[0240] The term "non-natural amino acid-based conjugation method" refers to a method of introducing non-genetically encoded amino acids into peptides or proteins, thereby introducing an active group, and preparing conjugates based on the active group. The non-natural amino acids are located at selected positions in the antibody peptide chain, and these positions are identified as optimal sites for substitution with non-natural amino acids. Each site is capable of carrying a non-natural amino acid with optimal structure, function, and / or antibody production. Some non-natural amino acids have side chains or functional groups that promote direct or synergistic conjugation of the antibody with a second reagent. In some embodiments, the antibody may exhibit enhanced conjugation efficiency, increased conjugation yield, or enhanced conjugation specificity compared to antibodies that do not have the same or other non-natural amino acids at other positions.

[0241] The term "Fc affinity peptide-based coupling method" refers to a method that specifically binds to a polypeptide or its derivative that binds to the Fc fragment, and then covalently modifies or directly loads the conserved amino acids of the Fc fragment. This includes, but is not limited to, Ajicap technology, MATE technology, and Fc peptides containing photoactivated amino acid residues (referring to non-naturally occurring UV-activated cross-linked amino acids within the peptide, such as benzoylphenylalanine (BPA). The peptide or its derivatives have a high affinity for the Fc moiety, for example, being or containing FcIII (DCAWHLGELVWCT) or FcIII 4c (CDCAWDLGELVWCTC), wherein the two cysteine ​​residues can form disulfide bonds found in natural proteins. In some embodiments, a selected position of the target agent is used for conjugation; for example, K246 or K248 (EU number) of the antibody agent is the conjugation position. In some embodiments, the conjugation position is K246 of the heavy chain. In some embodiments, the conjugation position is K248 of the heavy chain. In some embodiments, the conjugation position is K288 or K290 of the heavy chain. In some embodiments, the conjugation position is K288 of the heavy chain. In some embodiments, the conjugation position is K290 of the heavy chain. In some embodiments, the conjugation position is K317.

[0242] The term "glutaminase" refers to a series of enzymes in living organisms that catalyze the covalent transfer of alkylamines to glutamine (EC 2.3.2.13). The term "TGase" is used interchangeably with "glutaminase" herein. Some glutaminases known in the art can be used to prepare antibody-drug conjugates, including but not limited to glutaminases derived from various living organisms, such as TGases derived from guinea pig liver (GTGase), fish liver (FTGase), and microorganisms (mTGase), and any recombinant TGase (rTGase). Microbial glutaminases include, for example, TGases from *Streptomyces mobaraense*, *Streptomyces cinnamoneum*, and *Streptomyces griseocarneum*, from *Streptomyces lavendulae*, from *Bacillus subtilis*, and from *Streptomyces ladakanum*. An exemplary TGase is bacterial transglutaminase (BTG) (see, for example, EC 2.3.2.13, protein-glutamine-γ-glutamyltransferase). Glutaminases can also be mutant TGases (e.g., engineered) with at least 80% sequence homology to the natural TGase, such as T5398 and its mutants.

[0243] The term "formylglycine synthase" refers to a series of enzymes in organisms that can convert cysteine ​​or serine residues in the sulfatase motif to 2-formylglyine residues (referred to herein as "FGly") (EC 1.8.3.7). Although technically incorrect, the FGly residues produced by FGE are commonly referred to as "formylglyine" in the literature. The term "FGE" is used interchangeably with "formylglycine synthase" herein. Generally, enzymes that convert cysteine ​​(C) in the sulfatase motif to FGly are called FGEs; enzymes that convert serine (S) in the sulfatase motif to FGly are called Ats. In this document, "FGE" is generally used to refer to both types of FGly synthases, and it should be understood that the sulfatase motif (i.e., containing C or S) is present in the modified carrier protein. Some formylglycine synthases known in the art can be used to prepare antibody-drug conjugates, including but not limited to, for example, GenBank accession number NP_215226.

[0244] The term "isoprenetransferase" refers to a series of enzymes in organisms that catalyze the transfer of isopentenyl groups to receptors. These enzymes recognize certain amino acid motifs at or near the C-terminus of proteins and selectively alkylate them by adding (multiple) isoprene-like units to the protein carrying the motif at the sulfhydryl sites of (multiple) cysteine ​​residues of the ... FTase and GGTase I recognize the CAAX motif, while GGTase II recognizes the XXCC, XCXC, or CXX motif, where C represents cysteine, A represents an aliphatic amino acid, and X represents an amino acid that determines the substrate specificity of the isoprenoid transferase. Isoprene transferases from various sources can be used, such as those from humans, animals, plants, bacteria, and viruses. In some embodiments, naturally occurring or artificially modified isoprenoid transferases can be used. Examples include isoprenoid transferases with at least one naturally altered (including post-translational modifications) amino acid sequence, naturally occurring isoprenoid transferases in natural or artificially truncated forms, and modified isoprenoid transferases.

[0245] The term "transpeptidase" refers to a series of enzymes with transpeptidase activity in organisms, also known as sorting enzymes. Transpeptidases include, but are not limited to, sorting enzyme A (EC 3.4.22.70) and sorting enzyme B (EC 3.4.22.71). They have been found in various Gram-positive bacteria, such as Staphylococcus, Streptococcus, and Pneumococcus. Sorting enzyme A of the Gram-positive bacterium Staphylococcus aureus was first characterized. One of the advantageous features of sorting enzymes is that two molecules to be bound can require only a short peptide label ("sorting enzyme label"), which for Staphylococcus aureus is, for example, an LPTTG at the C-terminus of one molecule and a short chain of 3 to 5 amino acids of glycine at the N-terminus of another molecule. These peptide labels can be fused or bound to molecules via conventional cross-linking chemistry. This system allows for the linking of two proteins and can also be used to bind small molecular weight compounds, preferably small molecular weight toxin proteins. For example, the sorting enzyme B of Staphylococcus aureus has the NPQTN motif.

[0246] The term "sugar" refers to a polysaccharide or an oxidized or unoxidized molecule containing carbohydrates, including but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. In this document, "sugar" also refers to the carbohydrate portion of a sugar conjugate, such as glycoproteins, glycolipids, glycopeptides, glycoproteins, peptidoglycans, lipopolysaccharides, or proteoglycans. The term "glycan chain" is used interchangeably with "sugar" in this document.

[0247] As used herein, a "glycan linker" as an activated donor molecule for glycosyl coupling can be a synthetic sugar containing oxazoline or thiazoline, such as an oligosaccharide with a reduced-terminus activated, preferably an oligosaccharide molecule structured with oxazoline; it can also be a natural N-glycan oxazoline. Glycan linkers can also be chemically modified, for example, by introducing functional groups through azidation, alkylation, aldehyde sizing, thiolation, etc. Exemplary glycan linkers known in the art, including but not limited to those described in, for example, CN109071630B, WO2022050300A1, WO2023051814A1, etc., are incorporated herein by reference in their entirety and for all purposes. The term "disaccharide linker" is a glycan linker containing at least two monosaccharide units, preferably the two monosaccharide units linked by a glycosidic bond or a thioether bond. The term "disaccharide conjugate" refers to a disaccharide linker that further includes a payload such as a cytotoxin, and optionally also includes a linker between the disaccharide linker and the payload. Exemplary glycan linkers known in the art, including but not limited to those described, for example, in WO2025148977A1, WO2025038915A2, US12139509B2, CN114949236B, and WO2023232144A1, are incorporated herein by reference in their entirety and for all purposes.

[0248] The term "beta-N-acetylglucosinolate endonuclease" refers to a class of enzymes (EC 3.2.1.96) produced by a range of organisms that possess N,N'-diacetylchitobiosyl glycoside endonuclease activity. These enzymes typically belong to glycoside hydrolases 18 or 85. The term "ENGase" is used interchangeably with "beta-N-acetylglucosinolate endonuclease" herein. Some beta-N-acetylglucosinolate endonucleases known in the art, such as Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo Si, and Endo Se2, are described in WO2022 / 050300. Endo Si (also known as Endo-Si) is described in SEQ ID NO:49 of WO2024048490A1, and Endo Se2 (also known as Endo-Sz) is described in SEQ ID NO:55 of WO2024048490A1. These enzymes may also possess disaccharide linker transfer activity. Enzymes with this activity known in the art include Endo S, Endo S2, Endo F3, Endo Si, Endo Se2, and their mutants, as described in US12139509B2 and WO2025148977A1. Mutants of these enzymes may possess complex glycan linker transfer activity. Enzymes with this activity known in the art include mutants of Endo S, Endo S2, Endo F3, Endo Si, and Endo M, including but not limited to those described in CN109071630B and WO2022050300A1.

[0249] The term "galactosyltransferase" refers to a class of enzymes produced by a range of organisms that catalyze the transfer of galactose (EC 2.4.1.22 / 38 / 90). The terms "galactosyltransferase" and "GalT" are used interchangeably herein, and some galactosyltransferases known in the art can be used to prepare antibody-drug conjugates, and can be wild-type galactosyltransferases or mutants thereof. In some embodiments, the galactosyltransferase is a wild-type galactosyltransferase, preferably wild-type 3(1,4)-galactosyltransferase or wild-type (1,3)-N-galactosyltransferase. When the catalyst is a wild-type (1,3)-N-galactosyltransferase, human β(1,3)-Gal-T5 is preferred. The galactosyltransferase can be, for example, a mutant of β(1,4)-Gal-T1, such as a galactosyltransferase containing a mutant catalytic domain, etc. In some embodiments, the galactosyltransferase is an enzyme comprising a GalT mutant catalytic domain selected from Y289L, Y289N, Y289I, Y284L, R228K, Y289F, Y289M, Y289V, Y289G, and Y289A, preferably selected from Y289L, Y289N, Y289I, Y284L, and R228K.

[0250] The term "α-L-fucosidase" refers to a class of enzymes produced by a range of organisms that catalyze the hydrolysis of α-L-fucosides (EC 3.2.1.51). Some α-L-fucosidases known in the art can be used to prepare antibody-drug conjugates, such as α1,6-fucosidase (AlfC) from Lactobacillus casei or its mutants, including D200 or E274 mutations, including D200G, D200S, D200A, and D200S; and including E274A, E274S, E274G, and E274D.

[0251] The term "drug loading" (also expressed as DAR, DAR value, drug:antibody ratio) refers to the average amount of drug loaded per peptide-drug conjugate molecule in a peptide-drug conjugate population, or the ratio of drug amount to peptide amount. Drug loading can range from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conjugates per peptide. In some embodiments, the drug loading can be exemplarily 1, 2, 3, 4, 5, 6, or the average of any two values. The average amount of drug per ADC molecule after conjugation can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, CE-SDS assay, and HPLC characterization. Using the conjugation methods described herein, the first and second peptides typically have different numbers and / or types of drug conjugates. In this article, "DAR a+b" is used to describe the drug loading of dual-load conjugates, where "a" and "b" refer to the drug loading of the first and second polypeptide conjugates in the heterodimeric polypeptide conjugate, respectively. a or b can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0252] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0253] The term "treatment and / or prevention" refers to clinical interventions in the process of attempting to alter an individual's or treat a disease caused by cells, which can be preventative or interventional in a clinicopathological process. The effects of treatment and / or prevention include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and mitigating or improving prognosis.

[0254] The term "reducing conditions" or "reducing environment" refers to conditions or environments in which the substrate is more likely to be reduced than to be oxidized. The term "disulfide isomerization" refers to the interchange of disulfide bonds between different cysteine ​​residues, i.e., a reorganization of disulfide bonds. The minimum requirements for cysteine ​​residues in the hinge region to undergo disulfide isomerization can vary depending on the homodimer initiating protein, particularly on the exact sequence within the hinge region. Importantly, the homodimer interactions between the first and second Fc regions must be weak enough to allow cysteine ​​residues in the hinge region to undergo disulfide isomerization under given conditions.

[0255] In some embodiments, the reduction conditions can be achieved by adding a reducing agent, such as a reducing agent selected from the following: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP) or a chemical derivative thereof, L-cysteine ​​and β-mercaptoethanol, preferably a reducing agent selected from the following: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine or a chemical derivative thereof.

[0256] In some implementations, conditions are restored to a non-reducing or less reducing state, for example, by removing the reducing agent, such as through desalination.

[0257] In some embodiments, a method is used to prepare a first polypeptide or a second polypeptide using conventional techniques, conjugate it with a load, and then incubate it to obtain a heterodimeric polypeptide conjugate. This includes, for example, the following steps:

[0258] a) Provide a first polypeptide conjugate containing a first Fc region or a first polypeptide modified by coupling containing a first Fc region, and provide a second polypeptide conjugate containing a second Fc region, a second polypeptide modified by coupling containing a second Fc region or a second polypeptide containing a second Fc region.

[0259] The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region respectively.

[0260] b) Co-incubating a first polypeptide conjugate or a first polypeptide modified by coupling, and a second polypeptide conjugate, a second polypeptide modified by coupling, or a second polypeptide in the same reaction system; and

[0261] c) Obtain the heterodimeric polypeptide conjugate.

[0262] In some implementations, the first / second Fc region, the first / second polypeptide, the coupled first / second polypeptide, the first / second polypeptide conjugate, and the heterodimeric polypeptide conjugate are as defined above.

[0263] In some embodiments, the preparation method includes: firstly separating the homodimers of the first Fc region and the second Fc region; after the homodimer separation, because the heterodimer interaction between the first Fc region and the second Fc region is stronger than the homodimer interaction between the first Fc region and the second Fc region, the first Fc region and the second Fc region preferentially pair up and reassemble to form a heterodimeric polypeptide conjugate or a heterodimeric polypeptide. In some preferred embodiments, the method for separating the homodimers is to provide reducing conditions to open the disulfide bonds in the Fc region, and then provide oxidizing conditions to reassemble the heterodimers.

[0264] In some embodiments, step c) of the preparation method further includes removing the reducing agent.

[0265] In some embodiments, step c) of the preparation process further includes natural oxidation or the addition of an oxidizing agent.

[0266] In some embodiments, step c) further includes a purification step to remove the homodimer from the reaction system and purify the heterodimer polypeptide conjugate.

[0267] In some embodiments, this disclosure provides a first polypeptide conjugate containing a first Fc region and a second polypeptide conjugate containing a second Fc region, and incubates the first polypeptide conjugate and the second polypeptide conjugate to obtain a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

[0268] In some embodiments, this disclosure provides a first polypeptide conjugate comprising a first Fc region and a coupled-modified second polypeptide comprising a second Fc region, and incubates the first polypeptide conjugate and the coupled-modified second polypeptide to obtain a heterodimeric polypeptide conjugate with the first polypeptide coupled to a load. Further, a coupling reaction is performed on the obtained heterodimeric polypeptide conjugate with the coupled-modified second polypeptide and the load to obtain a heterodimeric polypeptide conjugate with both the first polypeptide and the second polypeptide coupled to a load.

[0269] In some embodiments, this disclosure provides a first polypeptide conjugate containing a first Fc region and a second polypeptide containing a second Fc region, and incubates the first polypeptide conjugate and the second polypeptide to obtain a heterodimeric polypeptide conjugate with the first polypeptide coupled to a load. Further, a coupling reaction between the second polypeptide and the load is performed on the obtained heterodimeric polypeptide conjugate to obtain a heterodimeric polypeptide conjugate with both the first polypeptide and the load coupled to a load; or, after modifying the obtained heterodimeric polypeptide conjugate with the second polypeptide to obtain a second polypeptide containing the coupled-modified second polypeptide, a coupling reaction is performed on the heterodimeric polypeptide conjugate with the coupled-modified second polypeptide and the load to obtain a heterodimeric polypeptide conjugate with both the first polypeptide and the load coupled to a load.

[0270] In some embodiments, this disclosure provides a first polypeptide comprising a first Fc region and a second polypeptide comprising a second Fc region, which are then coupled together to obtain a coupled heterodimeric polypeptide. Further, a coupling reaction is performed on the obtained heterodimeric polypeptide with the first polypeptide and / or the second polypeptide and a loading to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled together, or in which the first polypeptide is coupled together or the second polypeptide is coupled together. Even further, a coupling reaction is performed on the heterodimeric polypeptide conjugate in which the first polypeptide is coupled together with a loading to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled together; or, a coupling reaction is performed on the heterodimeric polypeptide conjugate in which the second polypeptide is coupled together with a loading to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled together. The payload can be coupled to both the first peptide and the second peptide simultaneously, either first to the first peptide and then to the second peptide, or first to the second peptide and then to the first peptide. The payload and coupling method are matched to the coupling positions of the coupled-modified first peptide and / or the coupled-modified second peptide.

[0271] In some embodiments, this disclosure provides a first polypeptide comprising a first Fc region and a second polypeptide comprising a second Fc region, which are then incubated to obtain a coupled heterodimeric polypeptide. Further, a coupling reaction between the first polypeptide and a load is performed on the obtained heterodimeric polypeptide conjugate to obtain a heterodimeric polypeptide conjugate; even further, a coupling reaction between the second polypeptide and a load is performed on the heterodimeric polypeptide conjugate containing the first polypeptide and a load to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled to the load. Further, a coupling reaction between the second polypeptide and a load is performed on the obtained heterodimeric polypeptide to obtain a heterodimeric polypeptide conjugate; even further, a coupling reaction between the first polypeptide and a load is performed on the heterodimeric polypeptide conjugate containing the second polypeptide and a load to obtain a heterodimeric polypeptide conjugate in which both the first and second polypeptides are coupled. Furthermore, a second peptide is modified onto the obtained heterodimeric polypeptide to obtain a heterodimeric polypeptide containing the coupled-modified second peptide; even further, a coupling reaction is performed on the obtained heterodimeric polypeptide with a first peptide and / or a second peptide and a loading to obtain a heterodimeric polypeptide conjugate. The loading can be coupled to both the first and second peptides simultaneously, either first to the first peptide and then to the second peptide, or first to the second peptide and then to the first peptide. The loading matches the coupling positions of the coupled-modified first and / or second peptides.

[0272] In some implementations, the loads, connectors, couplings, etc., are as defined above.

[0273] In some embodiments, the methods of this disclosure produce a product in which more than 80%, 85%, 90%, 95%, 99%, or 100% is the desired heterodimeric polypeptide conjugate.

[0274] In some embodiments, the product prepared by the method of this disclosure has an average DAR value of DAR1+1, DAR1+2, DAR1+3, DAR1+4, DAR2+2, DAR2+3, DAR2+4, DAR3+3, DAR3+4, or DAR4+4.

[0275] In some embodiments, the products prepared by the methods of this disclosure contain heterodimeric polypeptide conjugates of DAR1+1, DAR1+2, DAR1+3, DAR1+4, DAR2+2, DAR2+3, DAR2+4, DAR3+3, DAR3+4, or DAR4+4 in amounts exceeding 80%, 85%, 90%, 95%, 99%, or 100%.

[0276] 2. Example

[0277] In this disclosure, the packing material, chromatographic column, and instruments used for antibody, glycan-modified antibody, and ADC purification and property determination include: a 5 mL cOplete His-Tag Purification Column (Roche), and a HiLoad... TM 26 / 600 Superdex TM 200 prep grade column (Cytive), AmMag TM Protein A Magnetic Beads (GenScript), SDA030 Protein Purification System (Sepure), Acquity I-Class / RDa (Waters) Liquid Chromatography-Mass Spectrometer, Arc Premier High Performance Liquid Chromatography System, ACCQUITY UPLC BEH PROTEIN C4 (Waters, 1.7μm, 2.1mm×50mm) Column, TSKgel G3000SWXL (7.8mm×30cm, 5μm) SEC Column, TSKgel Butyl-NPR (4.6mm×10cm, 2.5μm) HIC Column.

[0278] In this disclosure, Ab1 / Ab2 were prepared according to patent WO2023246891A1, Ab3 / Ab4 were prepared according to patent WO2023083846A1, connector-load LP3 was prepared according to example B29 of patent WO2025087233A1, LP4 was prepared according to example D9 of patent WO2025167900A1, LP5 was prepared according to example 31 of patent WO2025103192A1, and LP7 was prepared according to example 35 of patent WO2025148977A1. Connectors-load LP6 (CAS: 2758671-45-1; item number: HY-148057), LP8 (CAS: 2129164-91-4; item number: HY-136314), and Deruxtecan (CAS: 1599440-13-7; item number: HY-13631E) were purchased from MedChemExpress. LLC. In this disclosure, the glycan linkers were commissioned to be synthesized by Wuhan Tangzhi Pharmaceutical Co., Ltd. Trastuzumab was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd. Other compounds and reagents, unless otherwise specified, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0279] Example 1: Fabrication of a dual-target, dual-load ADC

[0280] General preparation example

[0281] General Procedure 1: Antibody Preparation

[0282] The light and heavy chains of antibody Ab were cloned separately into the PTT5 expression vector to prepare transfection-grade expression plasmids. Expi293F cells were cultured in serum-free medium. TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C, 8% CO2, and 125 rpm. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. Feeding was performed on days 1 and 3 post-transfection. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and purified using a Protein A column for affinity purification. The column was washed with 1×PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0–3.5) and neutralized with 1M Tris-HCl (pH 8.0–9.0). The eluted sample was appropriately concentrated, replaced with 1×PBS, filtered, sterilized, and dispensed for use. The final purified antibody was analyzed by SDS-PAGE and HPLC purity and A280 concentration determination to confirm the availability of the antibody Ab.

[0283] General Procedure 2: Preparation Method of Non-Natural Sugar Engineered Antibodies

[0284] The antibody Ab, the prepared disaccharide linker, and ENGase (Endo Si or Endo Se2) were mixed in the same reaction system, with the concentrations of each being 10 mg / mL, 2 mM (30 times the antibody concentration equivalent), and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C for 6 h. After purification with protein A, the desired azide-modified non-natural sugar engineered antibody Ab was obtained.

[0285] General Operation 3: Site-directed conjugation ADC preparation method based on non-natural sugar engineered antibodies

[0286] The prepared azide-modified non-natural sugar engineered antibody Ab and cycloalkyne linker-loader were mixed to achieve concentrations of 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired sugar site-directed conjugation product ADC was obtained by protein A purification or ultrafiltration.

[0287] Compound Synthesis and Preparation Examples

[0288] Preparation Example 1: Preparation of Compound LP1

[0289] Step 1: In a 10 mL single-necked flask, add (2,5,8,11,14,17,20-heptaoxadecano-22-yloxy)acetic acid (1-1) (400.0 mg, 1.00 mmol) and HATU (380.0 mg, 1.00 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.25 mL, 2.00 mmol) to the resulting solution and stir the mixture at room temperature for 15 minutes. Then add compound 1-2 (460.2 mg, 1.00 mmol, prepared according to WO 2019208820) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify by preparative high-performance liquid chromatography to obtain compound 1-3.

[0290] LC-MS: 805.4 [M+H] + .

[0291] Step 2: Compound 1-3 (580.0 mg, 0.72 mmol) was added to a 50 mL single-necked flask and dissolved in TFA (2 mL) and dichloromethane (4 mL). The reaction solution was stirred at room temperature for 1 hour, then heated to 30 °C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to obtain compound 1-4.

[0292] LC-MS: 749.4 [M+H] + .

[0293] Step 3: Compound 1-5 (75.4 mg, 0.10 mmol), DIPEA (0.05 mL, 0.3 mmol), and compound 1-4 (74.9 mg, 0.10 mmol) were added to a 100 mL single-necked flask and dissolved in DMF (2 mL). DMTMM (28.3 mg, 0.10 mmol) was added to the resulting solution in an ice bath, and the reaction mixture was stirred in an ice bath for 1 hour. The reaction mixture was filtered and purified by high-performance liquid chromatography to obtain compound 1-6.

[0294] LC-MS: 1485.7 [M+H] + .

[0295] Step 4: Add compound 1-6 (100.0 mg, 0.07 mmol) to a 10 mL single-necked flask and dissolve in DMF (2 mL). Add diethylamine (0.07 mL, 0.71 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compound 1-7.

[0296] LC-MS: 1263.6 [M+H] + .

[0297] Step 5: Compounds 1-7 (80.0 mg, 0.06 mmol) and HATU (22.8 mg, 0.06 mmol) were added to a 10 mL single-necked flask and dissolved in DMF (2 mL). DIPEA (0.15 mL, 1.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Then, {1-(9H-fluorene-9-yl)-3-oxonyl-4-aza-2,7,10,13-tetraoxapentadecan-15-yl]oxy}acetic acid (28.4 mg, 0.06 mmol) was added to the mixture. The reaction mixture was evaporated to dryness, and the silica gel column was eluted with 5% methanol-dichloromethane solution to obtain 1-8.

[0298] LC-MS: 1718.9 [M+H] + .

[0299] Step 6: Add compounds 1-8 (50.0 mg, 0.03 mmol) to a 10 mL single-necked flask and dissolve in DMF (1 mL). Add diethylamine (0.45 mL, 0.3 mmol) to the resulting solution. Stir the reaction mixture at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, and slurry the residue with petroleum ether to obtain compounds 1-9.

[0300] LC-MS: 1496.8 [M+H]+ .

[0301] Step 7: In a 100 mL single-necked flask, add DBCO-acid (213.12 mg, 0.70 mmol), HATU (398.11 mg, 1.05 mmol), and HOBT (141.48 mg, 1.05 mmol), and dissolve in DMF (5 mL). Add DIPEA (0.23 mL, 1.40 mmol) to the resulting solution. Stir the mixture at room temperature for 5 minutes, then add compounds 1-9 (1044.69 mg, 0.70 mmol). Stir the reaction mixture at room temperature for 3 hours. Monitor the reaction for completeness using LCMS. After filtration, purify the reaction mixture using reverse-phase chromatography to obtain a pale yellow solid compound LP1.

[0302] LC-MS: 1783.3 [M+H] + .

[0303] 1 H NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 8.17 (d, J = 6.4Hz, 1H), 8.05 (d, J = 8.8Hz, 1H),7.88(d,J=8.8Hz,1H),7.80–7.73(m,2H),7.67–7.57(m,6H),7.51–7.42(m ,3H),7.38–7.28(m,6H),6.51(s,1H),5.45(s,2H),5.33–5.23(m,3H),5.08(s ,2H),5.02(d,J=14.4Hz,1H),4.41–4.35(m,2H),4.22–4.17(m,1H),3.91(s,2H ),3.83(s,2H),3.59–3.42(m,42H),3.30–3.26(m,2H),3.23(s,3H),3.14–3.0 3(m,5H),2.61–2.53(m,1H),2.37(s,3H),2.27–2.15(m,3H),2.02–1.95(m,2H) ,1.92–1.83(m,2H),1.79–1.72(m,1H),1.69–1.61(m,1H),1.58–1.50(m,1H),1 .42–1.36(m,2H),1.29(d,J=6.8Hz,3H),1.26–1.19(m,2H),0.90–0.81(m,9H).

[0304] Preparation Example 2: Preparation of Compound LP2

[0305] Reaction steps:

[0306] Step 1: Under ice bath conditions, 30 mL of dichloromethane was added to a 100 mL three-necked reaction flask, followed by the addition of compound undecylethylene glycol monomethyl ether propionic acid (3.0 g, 5.1 mmol), N-hydroxysuccinimide (615.8 mg, 5.35 mmol), and DCC (1.1 g, 5.35 mmol). The reaction mixture was stirred at room temperature for 2 hours until TLC (TLC) showed complete reaction of the starting materials. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain crude compound 2-1 (3.55 g, yield >100%). The crude product did not require purification and was used directly in the next step.

[0307] Step 2: Under nitrogen protection, compound Fmoc-L-lysine hydrochloride (1.6 g, 3.95 mmol) was added to 16 mL of dichloromethane, followed by compound 2-1 (2.98 g, 4.35 mmol) and triethylamine (1.1 mL, 7.9 mmol). The reaction mixture was stirred at room temperature for 3 hours until HPLC monitoring showed complete reaction of the starting materials. Citric acid monohydrate solution was added to the reaction mixture and stirred for 10 minutes. The mixture was separated, and the organic phase was collected. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1.9 g of crude product. This crude product was purified by reverse-phase chromatography (Spherical C18, 120 g; H2O / ACN: 5%-35%; flow rate: 60 mL / min). The fraction was collected and concentrated under reduced pressure to obtain compound 2-2 (942 mg, yield 25.4%).

[0308] Step 3: Compound 2-2 (600 mg, 638.91 μmol) and compound Val-Cit-PAB-MMAE (717.8 mg, 638.91 μmol) were added to DMF (6 mL), followed by TEA (133.6 μL, 958.36 μmol). The system was cooled in an ice bath and stirred for 10 minutes. Then, DMTMM (194.5 mg, 702.8 μmol) was added. The mixture was stirred at room temperature under nitrogen protection for 1 hour until HPLC monitoring showed complete reaction of the starting materials. The reaction solution was slowly added to an unsaturated NH4Cl aqueous solution to precipitate a solid. After stirring for 30 minutes, the mixture was filtered and dried under vacuum to obtain crude compound 2-3 (1.36 g, yield >100%). The crude product did not require purification and was used directly in the next step.

[0309] Step 4: Compound 2-3 (780 mg, 381.51 μmol) was added to DMF (7.8 mL), and diethylamine (395 μL, 3.81 mmol) was added dropwise under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours until HPLC monitoring showed that the starting material had reacted completely. The reaction solution was concentrated under reduced pressure, slurried with MTBE, filtered, and dried under vacuum to obtain crude compound 2-4 (695 mg, 100% yield). The crude product was used directly in the next step without further purification.

[0310] Step 5: Compounds 2-4 (695 mg, 381.39 μmol) were added to DMAc (7 mL). PPTS (95.6 mg, 381.39 μmol) was added first, and the mixture was stirred for 10 minutes. Then, BCN-PNP (144.2 mg, 457.67 μmol), HOBT (25.7 mg, 190.69 μmol), and TEA (106.5 μL, 762.77 μmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 hour until HPLC monitoring showed complete reaction of the starting materials. The reaction solution was purified by preparative liquid chromatography (C18, 30*150 nm, 5 μm; H2O / ACN: 10%-45%; flow rate: 20 mL / min). The fraction was collected to obtain 130 mg of compound LP2.

[0311] LC-MS (ESI) + )m / z 1000.3(M / 2+H) + .

[0312] ADC preparation example

[0313] Preparation Example 1: Preparation of Dual-Target Dual-Load ADC ddADC1

[0314] The heavy chain Ab1-EGFR-H and light chain Ab1-EGFR-L of antibody Ab1 targeting EGFR, and the heavy chain Ab2-B7H3-H and light chain Ab2-B7H3-L of antibody Ab2 targeting B7H3, were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids. Antibodies Ab1 and Ab2 were obtained through general operation one. Ab1-EGFR-H contains K370S, Y349T, and K409D substitutions, as shown in SEQ ID NO.1; the sequence of Ab1-EGFR-L is shown in SEQ ID NO.2; Ab2-B7H3-H contains S364R and D399K substitutions, as shown in SEQ ID NO.3; and the sequence of Ab2-B7H3-L is shown in SEQ ID NO.4.

[0315] Ab1-G23 and Ab2-G23 are non-natural sugar engineered antibodies obtained by combining the disaccharide linker G23 with antibodies Ab1 and Ab2 through general operation two. The structure of G23 is shown below.

[0316] Ab1-G23-LP1 is obtained from compound LP1 and the non-natural glycoengineered antibody Ab1-G23 via general operation three. The structure of LP1 is shown below. The measured full-length ADC HRMS value after deconvolution is 154553.47 Da (theoretical value is 154552.5 Da).

[0317] Ab2-G23-LP2 is obtained from compound LP2 and the non-natural glycoengineered antibody Ab2-G23 via general operation three. The structure of LP2 is shown below. The measured full-length ADC HRMS value after deconvolution is 154172.27 Da (theoretical value is 154176.16 Da).

[0318] By mixing Ab1-G23-LP1 and Ab2-G23-LP2, along with 2-mercaptoethylamine hydrochloride (2-MEA), to achieve final concentrations of 0.5 mg / mL, 0.5 mg / mL, and 10 mM, respectively, the reaction mixture was incubated at 37°C with a Tris-HCl buffer at a pH of 7.0–7.5 for 3 h on a shaker. The reducing agent was removed by ultrafiltration, and the solution was replaced with 1×PBS and allowed to oxidize naturally overnight at 4°C. After LC-MS confirmation of conversion to the product, the desired heterodimeric dual-load ddADC1 was obtained by purification with protein A or ultrafiltration.

[0319] LC-MS analysis revealed that the full-length ddADC1, after deconvolution, had a measured value of 154362.61 Da (the theoretical value is 154364.33 Da). These results demonstrate that a dual-target, dual-load ADC was successfully prepared using a conjugation method combined with in vitro recombinant antibody technology.

[0320] Preparation Example 2: Preparation of Dual-Target Dual-Load ADC ddADC2-5

[0321] The heavy chain Ab3-EGFR-H and light chain Ab3-EGFR-L of antibody Ab3 targeting EGFR, and the heavy chain Ab4-cMET-H and light chain Ab4-cMET-L of antibody Ab4 targeting cMET, were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids. Antibodies Ab3 and Ab4 were obtained through general operation one. Ab3-H contains F126C, C220V, T366W, and S354C substitutions, as shown in SEQ ID NO.5; Ab3-EGFR-L contains S121C and C214V substitutions, as shown in SEQ ID NO.6; Ab4-cMET-H contains T366S, L368A, Y407V, and Y349C substitutions, as shown in SEQ ID NO.7; and the sequence of Ab4-cMET-L is shown in SEQ ID NO.8.

[0322] Ab3-G23 and Ab4-G23 are non-natural sugar engineered antibodies obtained by combining disaccharide linker G23 with antibodies Ab3 and Ab4 through general operation two. Ab3-G7 and Ab4-G7 are non-natural sugar engineered antibodies obtained by combining disaccharide linker G7 with antibodies Ab3 and Ab4 through general operation two. The structure of G7 is shown below.

[0323] Ab3-G7-LP1 is obtained from compound LP1 and the non-natural glycoengineered antibody Ab3-G7 through general operation three. The measured value of the full-length ADC after deconvolution by HRMS is 149994.5 Da (theoretical value is 149987.96 Da).

[0324] Ab3-G23-LP1 is obtained from compound LP1 and the non-natural glycoengineered antibody Ab3-G23 through general operation three. The measured value of the full-length ADC after deconvolution by HRMS is 153610.9 Da (the theoretical value is 153603.64 Da).

[0325] Ab4-G7-LP2 is obtained from compound LP2 and the non-natural glycoengineered antibody Ab4-G7 through general operation three. The measured value of the full-length ADC after deconvolution by HRMS is 149570.3 Da (theoretical value is 149569.12 Da).

[0326] Ab4-G23-LP2 is obtained from compound LP2 and the non-natural glycoengineered antibody Ab4-G23 through general operation three. The measured value of the full-length ADC after deconvolution by HRMS is 153617.4 Da (theoretical value is 153617.06 Da).

[0327] ddADC2-5 was prepared by mixing Ab3-G7-LP1 or Ab3-G23-LP1 with Ab4-G7-LP2 or Ab4-G23-LP2 at a 1:1 molar ratio. The replacement buffer was 10 mM Tris-HCl buffer containing 100 mM sodium chloride, pH 7.5. After incubation at 37°C for 25 min, the mixture was cooled to 24°C and incubated for 30 min. Then, 2 mM dithiothreitol was added, and the mixture was incubated for 2 h. The antibody was concentrated to 10 mg / mL by ultrafiltration, and 5 mM dehydroascorbic acid was added, followed by incubation for 30 min. After LC-MS confirmation of conversion to the product, ddADC2-5 was obtained by purification with protein A or ultrafiltration. LC-MS detection data are shown in Table 1.

[0328] The results show that by using the conjugation method and in vitro recombinant antibody technology (modification of "node-acupoint" by antibody Fc), the DAR value of each load can be selected and precisely controlled at the same time as the preparation of dual-target dual-load ADC.

[0329] Table 1. Molecular weight detection of ddADC2-5

[0330] Preparation Example 3: Preparation of Dual-Target Dual-Load ADC ddADC6-12

[0331] The heavy chain Amivantamab-EGFR-H and light chain Amivantamab-EGFR-L of antibody Ab5, which targets EGFR, and the heavy chain Amivantamab-cMET-H and light chain Amivantamab-cMET-L of antibody Ab6, which targets cMET, were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids. Antibodies Ab5 and Ab6 were obtained through general operation one. Amivantamab-EGFR-H contains an F405L substitution, as shown in SEQ ID NO. 9, and the sequence of Amivantamab-EGFR-L is shown in SEQ ID NO. 10; Amivantamab-cMET-H contains a K409R substitution, as shown in SEQ ID NO. 11, and the sequence of Amivantamab-cMET-L is shown in SEQ ID NO. 12.

[0332] Ab5-G7 / G23 / G35 and Ab6-G7 / G23 / G35 are non-natural glycoengineered antibodies obtained by combining Ab5 and Ab6 with G7 / G23 / G35 through a general operation two. The structure of G35 is shown below.

[0333] Ab5-G7 / G23 / G35-LP2 and Ab6-G7 / G23 / G35-LP1 were obtained by using Ab5-G7 / G23 / G35 and Ab6-G7 / G23 / G35 with LP2 and LP1 respectively through general operation three. LC-MS detection data are shown in Table 2.

[0334] Table 2. Molecular weight detection of sugars using site-directed ADC based on Ab5 and Ab6.

[0335] By mixing Ab5-G7 / G23 / G35-LP2 and Ab6-G7 / G23 / G35-LP1, along with 2-mercaptoethylamine hydrochloride (2-MEA), the final concentrations of each were adjusted to 0.5 mg / mL, 0.5 mg / mL, and 75 mM, respectively. The reaction mixture was incubated at 37°C with a shaker for 3 hours using a Tris-HCl buffer system, adjusting the pH to 7.0-7.5. The reducing agent was removed by ultrafiltration, and the solution was replaced with 1×PBS and allowed to oxidize naturally overnight at 4°C. After LC-MS confirmation of product conversion, the desired heterodimeric dual-load ddADC6-12 was obtained by purification with protein A or ultrafiltration. ADC pairing and corresponding LC-MS detection data are shown in Table 3.

[0336] The results show that by using the coupling method and in vitro recombinant antibody technology (modification of antibody Fc charge pairing), dual-target dual-load ADCs can be prepared, and the DAR values ​​of each load can be selected and precisely controlled.

[0337] Table 3. Molecular weight detection of ddADC6-12

[0338] Preparation Example 4: Preparation of Dual-Load ADC dpADC1-8

[0339] The heavy chain Trastuzumab-H and light chain Trastuzumab-L of the HER2-targeting antibody Ab7-13 were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids. Antibody Ab7-13 was obtained through general procedure one. Specifically, Ab7-Trastuzumab-H contains D399R / S364H substitutions, as shown in SEQ ID NO.13; Ab8-Trastuzumab-H contains K409D / Y349T / K370S substitutions, as shown in SEQ ID NO.14; Ab9-Trastuzumab-H contains D399K / E356K substitutions, as shown in SEQ ID NO.15; Ab10-Trastuzumab-H contains K392D / K409D substitutions, as shown in SEQ ID NO.16; Ab11-Trastuzumab-H contains K370D / K409D substitutions, as shown in SEQ ID NO.17; and Ab12-Trastuzumab-H contains S364K / N390P / S400K substitutions, as shown in SEQ ID NO.13. As shown in NO.18, Ab13-Trastuzumab-H contains S364D / K370D / N390D / K392G / S400D substitutions, as shown in SEQ ID NO.19. The light chain sequences of the above antibodies Ab7-13 are all shown in SEQ ID NO:20.

[0340] Ab7 / 9 / 12-G35 is a non-natural glycoengineered antibody obtained by combining Ab7, Ab9, and Ab12 with G35 through general operation two. Ab7 / 9 / 12-G35-LP1 is obtained by combining Ab7 / 9 / 12-G35 with LP1 through general operation three.

[0341] Ab8-G7 / G23 / G35 are non-natural glycoengineered antibodies obtained by combining Ab8 with G7 / G23 / G35 through general operation two. Ab8-G7 / G23 / G35-LP2 is obtained by combining Ab8-G7 / G23 / G35 with LP2 through general operation three.

[0342] Ab13-G23 is a non-natural glycoengineered antibody obtained from Ab13 and G23 through general operation two. Ab13-G23-LP2 is obtained from Ab13-G23 and LP2 through general operation three.

[0343] Ab9-LP3 was obtained by random conjugation of Ab9 and LP3 via cysteine. Ab9 was reduced in 1×PBS with 14 equivalents of TCEP to a final concentration of 5 mg / mL at 37°C for 2 h. After reduction, the pH was adjusted to 6.0, followed by the addition of 12% DMSO. Then, 10 equivalents of LP3 were added at 25°C, and the reaction was incubated with shaking at 25°C for 1 h. After the reaction was completed, 100 mM N-acetyl-cysteine ​​solution was added at a ratio of 20:1 (quencher: antibody), and the mixture was shaken and quenched at room temperature for 20 min to terminate the conjugation reaction. The ADC was then transferred to PBS 7.4 buffer using desalting chromatography or ultrafiltration to obtain Ab9-LP3.

[0344] Ab10-LP4 was obtained by bridging Ab10 and LP4 via cysteine. Ab10 was reduced in 1×PBS with 10.5 equivalents of TCEP to a final concentration of 5 mg / mL at 37°C for 2 hours. After reduction, the pH was adjusted to 6.0, followed by the addition of 8% DMA. Then, 4.9 equivalents of LP4 were added, and the reaction was carried out at 25°C with shaking for 18 hours. After the reaction was completed, 100 mM N-acetyl-cysteine ​​solution was added at a quenching ratio of 20:1 (quencher:antibody), and the mixture was shaken and quenched at room temperature for 20 minutes to terminate the coupling reaction. The ADC was then replaced with 1×PBS buffer using desalting chromatography or ultrafiltration to obtain Ab10-LP4.

[0345] Ab11-LP5 was obtained by random conjugation of Ab11 and LP5 via cysteine. Ab11 was reduced in 1×PBS with 16 equivalents of TCEP to a final concentration of 5 mg / mL at 37°C for 2 h. Then, 12% DMSO was added, followed by 12 equivalents of LP5 at 25°C, and the reaction was incubated with shaking at 25°C for 1 h. After the reaction was complete, 100 mM N-acetyl-cysteine ​​solution was added at a 20:1 ratio (quencher: antibody), and the mixture was shaken and quenched at room temperature for 20 min to terminate the conjugation reaction. The ADC was then replaced with 1×PBS buffer using desalting chromatography or ultrafiltration to obtain Ab11-LP5.

[0346] The structures of LP3, LP4, and LP5 are shown below. The molecular weight determination data for Endo Si desugaring based on the Ab7-13 ADC are shown in Table 4.

[0347] Table 4. Molecular weight detection using a single-load ADC based on Ab7-13.

[0348] dpADC1-3 and 6-8 were prepared by mixing ADC-1 and ADC-2 (as shown in Table 5) with 2-mercaptoethylamine hydrochloride (2-MEA) to achieve final concentrations of 1.0 mg / mL, 1.0 mg / mL, and 25 mM, respectively. The reaction mixture was incubated at 37°C for 3 hours in a 1×PBS buffer system with the pH adjusted to 7.0-7.5. The reducing agent was removed by ultrafiltration, and the solution was replaced with 1×PBS and allowed to oxidize naturally overnight at 4°C. After LC-MS confirmation of product conversion, the desired heterodimeric dual loaders dpADC1-3 and 6-8 were obtained by purification with protein A or ultrafiltration. ADC pairing and corresponding LC-MS detection data are shown in Table 5.

[0349] dpADC4 was prepared by mixing ADC-1 and ADC-2 (as shown in Table 5) with 2-mercaptoethylamine hydrochloride (2-MEA) to achieve final concentrations of 1.0 mg / mL, 1.0 mg / mL, and 5 mM, respectively. The reaction mixture was incubated in a 1×PBS buffer system at 37°C for 3 hours with shaking to adjust the pH to 7.0-7.5. The reducing agent was removed by ultrafiltration, and the solution was replaced with 1×PBS and allowed to oxidize naturally overnight at 4°C. After LC-MS confirmation of conversion to the product, the desired heterodimeric dual-loador dpADC4 was obtained by purification with protein A or ultrafiltration. ADC pairing and corresponding LC-MS detection data are shown in Table 5.

[0350] dpADC5 was prepared by mixing ADC-1 and ADC-2 as shown in Table 5 to achieve final concentrations of 1.0 mg / mL and 1.0 mg / mL, respectively. The reaction mixture was incubated at room temperature for 16 h in a 1×PBS buffer system with the pH adjusted to 7.0-7.5. After LC-MS confirmation of the product, the desired heterodimeric dual-load dpADC5 was obtained by purification with protein A or ultrafiltration. ADC pairing and corresponding LC-MS detection data are shown in Table 5.

[0351] The results show that dual-load ADCs were prepared using cysteine-based random conjugation / bridging / sugar-based site-directed conjugation methods, and combinations thereof, in combination with in vitro recombinant antibody technology. Furthermore, the DAR values ​​of each load could be selectively and precisely controlled by choosing different conjugation methods.

[0352] Table 5. Molecular weight detection using dpADC1-8

[0353] Fabrication Example 5: Fabrication of Dual-Load ADC dpADC9

[0354] Route 1: Ab8-G27 is a non-natural glycoengineered antibody obtained from Ab8 and G27 through general operation 2. The structure of G27 is shown below. The measured value of Ab8-G27 after deconvolution in HRMS is 146816.9 Da (theoretical value is 146818.3 Da).

[0355] By mixing Ab7-G35-LP1 and Ab8-G27, along with 2-mercaptoethylamine hydrochloride (2-MEA), to achieve final concentrations of 1 mg / mL, 1 mg / mL, and 25 mM, respectively, the reaction mixture was incubated at 25°C for 16 h in a Tris-HCl buffer system with the pH adjusted to 7.0-7.5. The mixture was then subjected to ultrafiltration to remove the reducing agent, and the solution was replaced with 1×PBS and allowed to oxidize naturally overnight at 4°C. After LC-MS confirmation of conversion to the product, the desired LP1-conjugated heterologous recombinant molecule Ab7-G35-LP1 / Ab8-G27 was obtained via protein A purification or ultrafiltration. The measured value after HRMS deconvolution was 152247.8 Da (the theoretical value is 152241.5 Da).

[0356] The heterologous recombinant molecules Ab7-G35-LP1 / Ab8-G27 and the linker load LP6 were mixed to achieve concentrations of 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired dual-load ADC dpADC9 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 153727.5 Da (the theoretical value is 153721.4 Da).

[0357] Route 2: By mixing Ab7-G35 and Ab8-G27, and 2-mercaptoethylamine hydrochloride (2-MEA), the final concentrations of each were adjusted to 1 mg / mL, 1 mg / mL, and 25 mM, respectively. The reaction mixture was incubated at 25°C for 16 h using a Tris-HCl buffer system, with the pH adjusted to 7.0-7.5. The mixture was then incubated overnight at 4°C with the reducing agent removed by ultrafiltration. After confirmation of conversion to the product by LC-MS, the desired heterodimeric peptide Ab7-G35 / Ab8-G27 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 146890.3 Da (theoretical value 146889.5 Da).

[0358] The heterodimeric peptides Ab7-G35 / Ab8-G27, linker load LP1, and linker load LP6 were mixed to achieve concentrations of 5 mg / mL, 0.33 mM, and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired dual-load ADC dpADC9 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 153728.6 Da (the theoretical value is 153721.4 Da).

[0359] Route 3: By mixing Ab7 and Ab8-G27, and 2-mercaptoethylamine hydrochloride (2-MEA), the final concentrations of each were adjusted to 1 mg / mL, 1 mg / mL, and 25 mM, respectively. The reaction mixture was incubated at 25°C for 16 h in a Tris-HCl buffer system with the pH adjusted to 7.0-7.5. The mixture was then incubated overnight at 4°C with the reducing agent removed by ultrafiltration. After confirmation of conversion to the product by LC-MS, the desired LP1-conjugated heterologous recombinant molecule Ab7 / Ab8-G27 was obtained by purification with protein A or ultrafiltration. After deglycosylation with ENGase, the measured value after deconvolution by HRMS was 146435.4 Da (theoretical value 146433.1 Da).

[0360] The heterologous recombinant molecules Ab7 / Ab8-G27, G35, and Endo Si were mixed to achieve final concentrations of 10 mg / mL, 2 mM, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25 °C for 6 h. After LC-MS confirmation of conversion to the product, Ab7-G35 / Ab8-G27 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 146890.8 Da (the theoretical value is 146889.5 Da).

[0361] Ab7-G35 / Ab8-G27, linker load LP1, and linker load LP6 were mixed to achieve concentrations of 5 mg / mL, 0.33 mM, and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired dual-load ADC dpADC9 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 153728.6 Da (the theoretical value is 153721.4 Da).

[0362] The results showed that the glycogen-modified antibody could be recombined in vitro before being conjugated with a loading agent to prepare a dual-loading heterodimeric ADC. The order of the in vitro recombination and antibody conjugation steps could be adjusted.

[0363] Preparation Example 6: Preparation of Dual-Load ADC dpADC10

[0364] Route 1: By mixing Ab7-G35-LP1 and Ab8, and 2-mercaptoethylamine hydrochloride (2-MEA), the final concentrations of each were adjusted to 1 mg / mL, 1 mg / mL, and 25 mM, respectively. The reaction mixture was incubated at 25°C for 16 h in a Tris-HCl buffer system with the pH adjusted to 7.0-7.5. The mixture was then incubated overnight at 4°C with the reducing agent removed by ultrafiltration. After confirmation of conversion to the product by LC-MS, the desired LP1-conjugated heterologous recombinant molecule Ab7-G35-LP1 / Ab8 was obtained by purification with protein A or ultrafiltration. After desugaring with ENGase, the measured value after deconvolution by HRMS was 151656.8 Da (theoretical value 151648.8 Da).

[0365] Ab7-G35-LP1 / Ab8, linker load LP7, and Endo Si were mixed to achieve final concentrations of 10 mg / mL, 2 mM, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25 °C for 6 h. After LC-MS confirmation of conversion to the product, the desired dual-load ADC dpADC10 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 153718.0 Da (the theoretical value is 153711.3 Da).

[0366] Route 2: By mixing Ab7-G35-LP1 and Ab8, and 2-mercaptoethylamine hydrochloride (2-MEA), the final concentrations of each were adjusted to 1 mg / mL, 1 mg / mL, and 25 mM, respectively. The reaction mixture was incubated at 25°C for 16 h in a Tris-HCl buffer system with the pH adjusted to 7.0-7.5. The mixture was then incubated overnight at 4°C with the reducing agent removed by ultrafiltration. After confirmation of conversion to the product by LC-MS, the desired LP1-conjugated heterologous recombinant molecule Ab7-G35-LP1 / Ab8 was obtained by purification with protein A or ultrafiltration. After deglycation with ENGase, the measured value after deconvolution by HRMS was 151656.8 Da (theoretical value 151648.8 Da).

[0367] Ab7-G35-LP1 / Ab8, G40, and Endo Si were mixed to achieve final concentrations of 10 mg / mL, 2 mM, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25°C for 6 h. After LC-MS confirmation of conversion to the product, Ab7-G35-LP1 / Ab8-G40 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 152059.6 Da (the theoretical value is 152053.3 Da).

[0368] The Ab7-G35-LP1 / Ab8-G40 and LP8 linker were prepared at concentrations of 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired dual-loaded ADC dpADC10 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 153718.0 Da (the theoretical value is 153711.3 Da).

[0369] The results showed that a dual-loaded heterodimer ADC could be prepared by first recombining the first peptide with the second peptide in vitro, followed by conjugation with the second peptide conjugate. The conjugation with the second peptide conjugate could be performed by first modifying the second peptide and then conjugating it, or by directly conjugating it using a suitable method.

[0370] Fabrication Example 7: Fabrication of Dual-Load ADC dpADC11

[0371] Ab7 / Ab8-G27, LP7 linker, and Endo Si were mixed to achieve final concentrations of 10 mg / mL, 2 mM, and 0.4 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated at 25 °C for 6 h. After LC-MS confirmation of conversion to the product, the desired ADC Ab7-LP7 / Ab8-G27 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 148497.0 Da (the theoretical value is 148495.5 Da).

[0372] The Ab7-LP7 / Ab8-G27 and ligand LP6 were prepared at concentrations of 5 mg / mL and 0.33 mM, respectively. The pH of the reaction system was adjusted to 7.4, and the mixture was incubated overnight at 25°C. After LC-MS confirmation of conversion to the product, the desired ADC dpADC11 was obtained by purification with protein A or ultrafiltration. The measured value after deconvolution by HRMS was 149976.5 Da (the theoretical value is 149976.5 Da).

[0373] The results showed that after the modified first and second peptides were recombined, the second peptide could be coupled first, followed by the coupling of the first peptide, to prepare a dual-loaded heterodimer ADC.

[0374] As can be seen from the above preparation examples, the order of coupling of the first or second polypeptide, and the in vitro recombination of the first and second polypeptides, can be adjusted during the preparation of heterodimeric polypeptide conjugates. The preparation of heterodimeric conjugates can be achieved through in vitro recombination of the first and second polypeptide conjugates, or through in vitro recombination of the first and second polypeptide conjugates followed by conjugation of the second polypeptide conjugate; alternatively, it can be achieved through in vitro recombination of the first and second polypeptides (modified by coupling) followed by conjugation of the first and second polypeptides; or through in vitro recombination of the first and unmodified second polypeptide, followed by modification of the second polypeptide, and then simultaneous or sequential conjugation of the first and second polypeptides; or through in vitro recombination of the first and unmodified second polypeptide, followed by conjugation of the second polypeptide conjugate, and then conjugation of the first polypeptide conjugate.

[0375] Example 2 Analysis of Dual-Load ADC

[0376] The analysis method is as follows:

[0377] 1. Hydrophobic chromatography (HIC-HPLC)

[0378] Sample preparation: The sample was diluted to 3.0 mg / mL with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0379] Chromatographic conditions:

[0380] Gradient elution procedure:

[0381] 2. Size Exclusion Chromatography (SEC-HPLC)

[0382] Sample preparation: Sample concentration 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane; Instrumentation: Agilent 1260 Infinity II Bio-Inert LC System; Column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm; Mobile phase: 0.2 M phosphate buffer, 5-15% isopropanol pH 7.0; Flow rate: 0.5-1 mL / min; Detection wavelength: 280 nm & 360 nm; Column temperature: room temperature; Loading amount: 30 μg; SEC elution method: isocratic elution.

[0383] Preparation of the positive control molecule T-DXd: Trastuzumab was replaced with 10 mM phosphate buffer (pH 7.0) by desalting chromatography or ultrafiltration, and the antibody concentration was determined using a UV method. 10 mM TCEP solution (6-10 equivalents) was added to the antibody solution, mixed, and reduced at 37°C for 3 hours. The reaction solution was then cooled to 15-25°C in a water bath, and 10 mM dimethyl sulfoxide solution containing Deruxtecan (10-20 equivalents) was added, mixed, and reacted for 1.5-2 hours. After the conjugation reaction was complete, 100 mM N-acetylcysteine ​​solution was added at a quenching ratio of 20:1 (quencher:antibody), shaken well, and quenched at room temperature for 20 min to terminate the conjugation reaction. The ADC buffer was replaced with a 20 mM His-HAc buffer at pH 5.5 using desalting chromatography or ultrafiltration, and then filtered through a 0.22 μm PES filter to obtain T-DXd. The DAR value of HIC detection was 7.87, and the SEC monomer peak percentage was 98.64%.

[0384] The preparation of dpADC3 is described in Example 4 of ADC preparation. Mass spectrometry, HIC, and SEC were used for analysis. The mass spectrometry analysis is shown in Figure 5. No original ADC residue was observed after in vitro recombination of Ab7-G35-LP1 and Ab8-G35-LP2. The DAR value was 3+3. The main peak percentage in HIC was 91.33%, and the monomer peak percentage in SEC was 95.18%.

[0385] Therefore, in vitro recombination technology can be used to prepare dual-load ADCs efficiently and uniformly.

[0386] Example 3: In vivo efficacy of dual-load ADC

[0387] Using BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) as test animals, the efficacy of dual-loaded ADC administered via tail vein injection on nude mice with human gastric cancer cell NCI-N87 xenografts was evaluated.

[0388] Mice were subcutaneously inoculated with NCI-N87 cells in the right rib area (Source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (5×10⁻⁶) 6 / each with 50% artificial basement membrane), the tumor grew to 165.4±36.5 mm in 7 days. 3 The animals were then randomly grouped (dl) into groups of 5.

[0389] The drug was administered via a single tail vein injection at a dose of 2 mg / kg. Tumor volume and body weight were measured twice weekly, and the data were recorded. Tumor inhibition rate (%) = (Crtv - Trtv) / Crtv (%), where Crtv and Trtv were the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group, respectively, at the end of the experiment. The specific experimental results at the end of the experiment on day 21 after administration are shown in Figure 6. The tumor inhibition rates of the T-DXd group and the dpADC3 group were 60.0% and 107.6%, respectively.

[0390] The results showed that the dual-load ADC drug prepared by the method of this disclosure exhibited superior antitumor activity against HER2-overexpressing NCI-N87 xenografts compared to the positive control T-DXd.

[0391] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify various modifications and equivalents of a particular compound, material, and operation without the need for extraordinary testing. All such modifications and equivalents are within the scope of this disclosure and are encompassed by the appended claims.

Claims

1. A method for preparing a heterodimeric polypeptide conjugate, the method comprising the following steps: a) Provide: ① a first polypeptide conjugate, and ② a second polypeptide conjugate, or a second polypeptide modified by conjugation, or a second polypeptide; The first polypeptide contains a first Fc region, and the second polypeptide contains a second Fc region. The first polypeptide conjugate is coupled to the first polypeptide containing the first Fc region, and the second polypeptide conjugate is coupled to the second polypeptide containing the second Fc region. The second polypeptide modified by coupling is modified to have a different coupling position with the second polypeptide. The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region respectively. b) Co-incubation in the same reaction system of: ① a first polypeptide conjugate, and ② a second polypeptide conjugate, or a second polypeptide modified by conjugation, or a second polypeptide; and, c) Obtain the heterodimeric polypeptide conjugate.

2. The preparation method according to claim 1, wherein, After incubating the first polypeptide conjugate and the modified second polypeptide in step b) to obtain the heterodimeric polypeptide conjugate in step c), the process further includes the following steps: d) Add a load to cause the modified second polypeptide contained in the heterodimeric polypeptide conjugate to undergo a coupling reaction with the load, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load. or, After incubating the first polypeptide conjugate and the second polypeptide in step b) to obtain the heterodimeric polypeptide conjugate in step c), the process further includes the following steps: d) Add a load to cause the second polypeptide contained in the heterodimeric polypeptide conjugate to undergo a coupling reaction with the load, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load; or, further modify the second polypeptide in the heterodimeric polypeptide conjugate into a coupled-modified second polypeptide, and then add a load to cause the coupled-modified second polypeptide in the heterodimeric polypeptide conjugate to undergo a coupling reaction with the load, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load.

3. A method for preparing a heterodimeric polypeptide conjugate, the method comprising the following steps: a) Provide: ① a first polypeptide modified by coupling, and ② a second polypeptide modified by coupling, or a second polypeptide; The first polypeptide contains a first Fc region, and the second polypeptide contains a second Fc region. The first polypeptide, which has been coupled and modified, has a different coupling position with the first polypeptide. The second polypeptide, which has been coupled and modified, has a different coupling position with the second polypeptide. The first Fc region and the second Fc region have different sequences, which makes the heterodimer interaction between the first Fc region and the second Fc region stronger than the homodimer interaction between the first Fc region and the second Fc region respectively. b) Co-incubate in the same reaction system: ① the first polypeptide modified by coupling, and ② the second polypeptide modified by coupling, or the second polypeptide; c-1) Obtain the coupled-modified heterodimeric polypeptide; c-2) Add a load to cause it to undergo a coupling reaction with the first peptide and / or the second peptide contained in the coupled-modified heterodimeric polypeptide, thereby obtaining a heterodimeric polypeptide conjugate with a first peptide coupled load and / or a second peptide coupled load.

4. The preparation method according to claim 3, wherein after step b) incubating the first and second peptides modified by coupling, and after obtaining the heterodimeric peptide modified by coupling in step c-1), the second peptide contained in the heterodimeric peptide is further modified into a second peptide modified by coupling, and then the coupling reaction in step c-2) is carried out.

5. The preparation method according to claim 3, wherein step b) incubates the first and second peptides modified by coupling, step c-1) obtains the heterodimeric peptide modified by coupling, and step c-2) adds a load to cause it to undergo a coupling reaction with the first peptide modified by coupling contained in the heterodimeric peptide modified by coupling to obtain a heterodimeric peptide conjugate, and then the second peptide in the heterodimeric peptide conjugate is further modified into a second peptide modified by coupling.

6. The preparation method according to any one of claims 3 or 4, wherein after step c-2) adding a loading to cause it to undergo a coupling reaction with the second polypeptide contained in the coupled-modified heterodimeric polypeptide or the coupled-modified second polypeptide, the method further comprises the step of: d) Add a load to cause it to undergo a coupling reaction with the first peptide that has been coupled and modified in the heterodimeric peptide conjugate, thereby obtaining a heterodimeric peptide conjugate in which both the first peptide and the second peptide are coupled with loads.

7. The preparation method according to any one of claims 3-5, wherein: Step c-2) involves adding a load to couple it with the first coupled polypeptide contained in the coupled heterodimeric polypeptide to obtain a heterodimeric polypeptide conjugate. The process further includes the following steps: d) Add a load to make it couple with the second polypeptide contained in the heterodimeric polypeptide conjugate or the second polypeptide modified by coupling, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load. or, Step c-2) involves adding a load to couple it with the first polypeptide in the coupled heterodimeric polypeptide to obtain a heterodimeric polypeptide conjugate. The second polypeptide in the heterodimeric polypeptide conjugate is further modified into a coupled second polypeptide, and the process further includes the following steps: d) Add a load to cause it to couple with the modified second polypeptide contained in the heterodimeric polypeptide conjugate, thereby obtaining a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load.

8. The preparation method according to any one of claims 3-4, wherein in step c-2), a load is added to cause it to undergo a coupling reaction simultaneously with the first peptide and the second peptide, or the second peptide, contained in the coupled-modified heterodimeric polypeptide, thereby obtaining a heterodimeric polypeptide conjugate in which both the first peptide and the second peptide are coupled with loads.

9. The production process according to any one of claims 1 to 8, wherein The first polypeptide is a first antibody containing a first antigen / target binding fragment, or a first fusion protein containing a first Fc region. And / or, The second polypeptide is a second antibody containing a second antigen / target binding fragment, or a second fusion protein containing a second Fc region; Preferably, the first polypeptide and the second polypeptide bind to the same epitope of the same target, or different epitopes of the same target, or epitopes of different targets. More preferably, the target bound by the first polypeptide and / or the second polypeptide is selected from any of the following antigens or any combination thereof: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5, c-Met, HERV-capsule protein, periosteal protein, Big3, SPARC, BCR, CD79, CD37, EGFrRvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM, MRP3, TROP2, Nectin-4, CLDN18.2, CLDN6, CDH6, B7-H3, ROR1, PSMA, PD-L1, FRα, DLL3, CDH17, 5T4, and Tissue. Factor, PTK7, BCMA, NaPi2b, B7-H4, LIV-1, CEACAM5, CD22, CD33, MSLN, CD30, GPC3, IL3RA, FGFR2b, CDCP1, CD73, uPARAP, KIT, IL2RA, ICAM-1, GUCY2C, CDH3, CD79b, CD70, VEGFR2, VEGF, TFR1, S TEAP1, SEZ6, NCAM1, MUC16, LRRC15, IL13RA2, DLK1, CLL-1, CD45, CD37, BCL2L1, KIF11, ITGB6, IL1RAP, FLT3, FGFR2, EphA2, DPP-4, CD7, CD47, TRAILR2, TLR7, Tau, Tumor-associated carbohydrate antigen (Tumor-associated carbohydrate (antigen), SORT1, SLC1A5, SDC1, PD-1, NMT, NA+ / K+ATPASE, LY6E, IGF-1R, HSP70, glucocorticoid receptor (GR), FGFR3, ENPP3, CD105, CTLA4, CLDN1, CEACAM6, CD228, CD147, ADAM9, AMHR2, ALPP, ALPG or LY6G6D, etc.; and / or, CD3, CD4, CD8, CD40, CD25, CD28, CD16, CD89, CD32, CD64, FcεRI or CD1, etc.

10. The production process according to any one of claims 1 to 9, wherein The first Fc region is selected from IgG1, IgG2, IgG3 and IgG4 isotypes, and / or the second Fc region is selected from IgG1, IgG2, IgG3 and IgG4 isotypes; Preferably, both the first Fc region and the second Fc region are IgG1 or IgG4 isotypes.

11. The method of manufacture of any one of claims 1-10, wherein the first Fc region and / or the second Fc region contains an amino acid substitution, wherein, The amino acid substitution is selected from one or more amino acid substitutions based on charge interaction and / or amino acid substitutions based on non-electrostatic interaction; Preferably, the one or more charge-based amino acid substitutions are selected from one or more positively charged amino acids and negatively charged amino acids, or positively charged amino acids and uncharged amino acids, or negatively charged amino acids and uncharged amino acids. More preferably, the first Fc region and the second Fc region each contain amino acid substitutions selected from any one of the following groups, wherein the numbers are EU numbers: T366S / L368A / Y407V, T366W; S354C / T366W, Y349C / T366S / L368A / Y407V; T366Y, Y407T; T366W, Y407A; T394W, F405A; T366Y / F405A, T394W / Y407T; T366W / F405W, T394S / Y407A; F405W, T394S; D399C, K392C; T366W / D399C, T366S / L368A / K392C / Y407V; T366W / K392C, T366S / L368A / D399 C / Y407V; S354C / T366W, Y349C / T366S / L368A / Y407V; Y349C / T366W, S354C / T366S / L368A / Y4 07V; E356C / T366W, Y349C / T366S / L368A / Y407V; Y349C / T366W, E356C / T366S / L368A / Y407V; E357C / T366W, Y349C / T366S / L368A / Y407V; Y349C / T366W, E357C / T366S / L368A / Y407V; T366K , L351D; T366K, Y349E / D; T366K, L368E / D; T366K, L351D / Y349D / R355D; T366K, Y349E / D / L36 8E / D / L351D; T366K / L351K, L351D; T366K / L351K, Y349E / D; T366K / L351K, L368E / D; T366K / L 351K, Y349E / D / L368E / D / L351D; T366K / L351K, L351D / Y349D / R355D; L351K, L351D; K370E / D 399K / K439D, D356K / E357K / K409D; K409D, D399K; K409E, D399K; K409E, D399R; K409D, D399R; D399K, K370T; S364R / D399K / E357N, K370S / Y349T / Q347D / K409D; S364R / D399K, K370S / Y349T / Q347D / K409D; F405L, K409R; WT, F405L / R409K; T350I / K370T / F405L, K409R; K370W, K409R; D399A / F / G / H / I / L / M / N / R / S / T / V / W / Y, K409R; T366A / D / E / F / G / H / I / L / M / Q / V / Y, K409R; L368A / D / E / G / H / N / R / S / T / V / Q, K409A / G / R / H; D399F / H / K / R / Q, K409A / G / R / H; F405I / K / L / S / T / V / W, K409A / G / R / H; Y407L / W / Q, K409A / G / R / H; D360K, K402D; D403K, K419D; E357K, K370E; K249E / K288E, E236K / D278K; H435R, WT; S364H, Y349T; S364H / F405A, Y349T / T394F; S364H, Y349T; D399R / S364H, K409D / Y349T / K370S; D399K / S364H, K409D / Y349T / K370S; D399K / E356K, K370D / K409D;S364K / N390P / S400K, S364D / K370D / N390D / K392G / S400D; More preferably, the first Fc region and the second Fc region each contain amino acid substitutions selected from any one of the following groups, wherein the numbers are EU numbers: D399R / S364H, K409D / Y349T / K370S; D399K / S364R, K409D / Y349T / K370S; D399K / S364H, K409D / Y349T / K370S; D399K / E356K, K392D / K409D; D399K / E356K, K370D / K409D; S364K / N390P / S400K, S364D / K370D / N390D / K392 G / S400D; D399K / E357K, K392D / K409D; D399K / E357K, K370D / K409D; F40 5L, K409R; WT, F405L / R409K; T366S / L368A / Y407V / Y349C, T366W / S354C.

12. The method of producing according to any one of claims 9-11, wherein the first polypeptide comprises a first heavy chain H1 and a first light chain L1; and / or, the second polypeptide comprises a second heavy chain H2 and a second light chain L2; wherein, H1 and H2 contain the CH1 structural domain, and L1 and L2 contain the CL structural domain; Preferably, the first polypeptide and / or the second polypeptide improve the correct pairing of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 through technologies such as CrossMab, DuetMab, common light chain, and common heavy chain. Preferably, the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 comprise: (a) a modified CH1 domain comprising a substitution of a naturally occurring noncysteine ​​amino acid to cysteine; and (b) a modified corresponding CL domain comprising a substitution of a naturally occurring noncysteine ​​amino acid to cysteine. More preferably, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following substitutions, wherein the EU numbers are as follows: CH1: the amino acid at position 126 is substituted with cysteine, and the cysteine ​​at position 220 is substituted with a non-cysteine ​​amino acid; CL: the amino acid at position 121 is substituted with cysteine, and the cysteine ​​at position 214 is substituted with a non-cysteine ​​amino acid. More preferably, the CH1 and CL domains of the first heavy chain H1 and the first light chain L1 and / or the second heavy chain H2 and the second light chain L2 respectively contain the following substitutions, wherein the EU numbers are: CH1: F126C / C220V, CL: S121C / C214V.

13. The preparation method according to any one of claims 1-8, wherein the first polypeptide is coupled with one or more types of loads, each type of load containing one or more; and / or the second polypeptide is coupled with one or more types of loads, each type of load containing one or more; wherein, The loadings of the first peptide conjugate and the second peptide conjugate may be exactly the same, partially the same, or different in type and / or number.

14. The production method according to claim 13, wherein The payload is a cytotoxin, a small molecule drug, a near-infrared or fluorescent probe, a peptide, a nucleic acid and related drugs, a radioactive isotope label, a contrast agent, or a magnetic resonance imaging agent, etc.; the small molecule drug is preferably selected from maytansine, DM-1, DM-4, ​​MMAE, MMAF, Auristatin 0101, SN-38, DXd, exatecan, docarmycin, amanitin, PBDs, VP-16, camptothecin, paclitaxel, docetaxel, anthracyclines, and derivatives of the above compounds, or the small molecule drug is a radiotherapy agent; Preferably, the first polypeptide and the second polypeptide are coupled with different types of payloads.

15. The production method according to claim 13 or 14, wherein The first polypeptide is conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 payloads; and / or the second polypeptide is conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 payloads; Preferably, the first polypeptide is coupled with 1, 2, 3, 4, 5 or 6 load cells; and / or the second polypeptide is coupled with 1, 2, 3, 4, 5 or 6 load cells; Preferably, the first polypeptide and the second polypeptide are coupled with different numbers of payloads.

16. The preparation method according to any one of claims 1-8, wherein the first polypeptide-coupled loading or the second polypeptide-coupled loading is prepared by fusion expression, random coupling, or site-directed coupling methods. wherein, The first polypeptide-coupled load and the second polypeptide-coupled load are prepared by a combination of one or more coupling methods. The preparation methods of the first polypeptide-coupled load and the second polypeptide-coupled load can be the same, partially the same or different. The loads are coupled at the same site, partially the same site or different site of the first polypeptide and the second polypeptide. Preferably, the random coupling method is a lysine-based random coupling method, a cysteine-based random coupling method, or a bridging method. Preferably, the site-directed coupling method is a site-directed coupling method based on non-natural amino acids, a site-directed coupling method based on engineered cysteine ​​mutant antibodies, a site-directed coupling method based on enzymes, a site-directed coupling method based on Fc affinity peptides, or a site-directed coupling method based on sugars.

17. The preparation method of claim 16, wherein the random coupling method is based on a cysteine ​​coupling method or a bridging method.

18. The preparation method of claim 16, wherein the site-directed coupling method is an enzyme-based site-directed coupling method or a sugar-based site-directed coupling method, wherein, Enzyme-based site-directed coupling methods are selected from coupling methods based on the following enzymes and their mutants, or modified enzymes containing their catalytic domains: sortase, prenyltransferase, formylglycine-generating enzyme, transglutaminase, spyLigase, horseradish peroxidase, tyrosinase, phosphopantetheinyl transferase, etc.; and / or The sugar-based site-directed coupling method is selected from the coupling method based on glycosylation, or the coupling method based on the following enzymes and their mutants, or modified enzymes containing their catalytic domains: galactosyltransferase (GalT), beta-N-acetylglucosinolate endonuclease (ENGase), α-L-fucosidase, etc.

19. The preparation method according to claim 18, wherein the glutamine transferase is selected from microbial transglutaminase (mTGase) and its mutants, or modified enzymes containing its catalytic domain; The galactosyltransferase is selected from β(1,4)-Gal-Tl and its mutants, or modified enzymes containing its catalytic domain; The beta-N-acetylglucosinolate endonuclease is selected from Endo Si, Endo Se2, Endo S, Endo S2, Endo F3, Endo M and their mutants, or modified enzymes containing their catalytic domain.

20. The preparation method according to any one of claims 16-19, wherein the first polypeptide and the second polypeptide are independently coupled with the load by means selected from glycosite coupling, random coupling, and / or bridging.

21. The preparation method according to claim 17 or 20, wherein the first polypeptide and / or the second polypeptide couple the loading to the conserved cysteine ​​of the polypeptide via a thiol reactive group such as maleimide, 2-methylsulfonyl pyrimidine, haloacetamide, etc. Preferably, the connector is a bridging connector.

22. The preparation method of claim 21, further comprising the step of coupling the first polypeptide and / or the second polypeptide to a load via a thiol reactive group; Preferably, the preparation method includes mixing and incubating a first polypeptide and / or a second polypeptide with a reducing agent, adding a loading agent to react, and purifying to obtain a first polypeptide conjugate and / or a second polypeptide conjugate. More preferably, the preparation method includes adding an organic solvent; More preferably, the preparation method includes adding a quencher to terminate the reaction.

23. The preparation method according to any one of claims 18-20, wherein the first polypeptide and / or the second polypeptide are coupled to the enzymatic reaction site of the polypeptide via a linker in an enzymatic manner; Preferably, the linker is a disaccharide linker or a complex glycan linker, and the load is coupled at the conserved N297 N-glycosylation modification site.

24. The preparation method of claim 23, further comprising the step of coupling the first polypeptide and / or the second polypeptide via a disaccharide linker, ENGase and load; Preferably, the first polypeptide and the second polypeptide are mixed with a disaccharide linker and an ENGase, respectively, and incubated to obtain the desired non-natural sugar engineered first polypeptide or non-natural sugar engineered second polypeptide; optionally, the non-natural sugar engineered first polypeptide and the non-natural sugar engineered second polypeptide are then mixed with a load, respectively, and incubated to obtain a first polypeptide conjugate or a second polypeptide conjugate. Preferably, incubating the first polypeptide conjugate and the second polypeptide conjugate yields a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a loading. Preferably, after incubating the first polypeptide conjugate and the non-natural sugar engineered second polypeptide, or obtaining a heterodimeric polypeptide conjugate from the second polypeptide, the heterodimeric polypeptide conjugate and the load are mixed, and after incubation, a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with the load is obtained. Preferably, after incubating the first polypeptide conjugate and the second polypeptide to obtain a heterodimeric polypeptide conjugate, the heterodimeric polypeptide conjugate is mixed with a disaccharide linker and ENGase, and after incubation, a non-natural sugar engineered heterodimeric polypeptide conjugate modified with the second polypeptide is obtained; optionally, the non-natural sugar engineered heterodimeric polypeptide conjugate modified with the second polypeptide is then mixed with a load, and after incubation, a heterodimeric polypeptide conjugate in which both the first polypeptide and the second polypeptide are coupled with a load is obtained.

25. The preparation method of claim 23, further comprising the step of coupling the first polypeptide and / or the second polypeptide via a disaccharide linker, ENGase and a load; wherein the first polypeptide and the second polypeptide are respectively mixed with the disaccharide linker and ENGase, and incubated to obtain the desired non-natural sugar engineered first polypeptide or non-natural sugar engineered second polypeptide; Preferably, a non-natural sugar engineered first polypeptide and a non-natural sugar engineered second polypeptide are incubated to obtain a non-natural sugar engineered heterodimer polypeptide in which both the first polypeptide and the second polypeptide are modified. More preferably, the non-natural sugar engineered heterodimeric peptide and the load are mixed and incubated to obtain a heterodimeric peptide conjugate in which both the first peptide and the second peptide are coupled with the load. Preferably, the first and second non-natural sugar engineered polypeptides are incubated to obtain a non-natural sugar engineered heterodimer polypeptide modified from the first polypeptide; More preferably, the non-natural sugar engineered heterodimeric peptide modified with the first peptide is mixed with the load, and after incubation, a heterodimeric peptide conjugate in which both the first peptide and the second peptide are coupled with the load is obtained. More preferably, the non-natural sugar engineered heterodimeric peptide modified with the first peptide is mixed with a disaccharide linker and ENGase, and incubated to obtain a non-natural sugar engineered heterodimeric peptide modified with both the first peptide and the second peptide; optionally, the non-natural sugar engineered heterodimeric peptide is then mixed with a load, and incubated to obtain a heterodimeric peptide conjugate in which both the first peptide and the second peptide are coupled with the load. More preferably, the non-natural sugar engineered heterodimeric peptide modified with the first peptide is mixed with the load and incubated to obtain a heterodimeric peptide conjugate with the second peptide coupled with the load; optionally, the heterodimeric peptide conjugate is then mixed with the load and incubated to obtain a heterodimeric peptide conjugate with both the first peptide and the second peptide coupled with the load. More preferably, the non-natural sugar engineered heterodimeric peptide modified with the first peptide is mixed with the load and incubated to obtain a heterodimeric peptide conjugate with the first peptide coupled with the load; optionally, the heterodimeric peptide conjugate is then mixed with the load and incubated to obtain a heterodimeric peptide conjugate with both the first peptide and the second peptide coupled with the load. More preferably, the first polypeptide is modified into a non-natural sugar-engineered heterodimeric polypeptide and a load is mixed and incubated to obtain a heterodimeric polypeptide conjugate with the first polypeptide coupled to the load; optionally, the first polypeptide-coupled heterodimeric polypeptide conjugate is then mixed with a disaccharide linker and ENGase, and incubated to obtain a second polypeptide-modified heterodimeric polypeptide conjugate; optionally, the heterodimeric polypeptide conjugate is then mixed with the load, and incubated to obtain a heterodimeric polypeptide conjugate with both the first polypeptide and the second polypeptide coupled to the load.

26. The method of making according to claims 1-8, wherein, Step b) further includes: incubating the first polypeptide together with the second polypeptide under reducing conditions sufficient to allow the cysteine ​​in the hinge region to undergo disulfide bond isomerization; Preferably, the reduction conditions include the addition of a reducing agent selected from the following: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine or its chemical derivatives, dithioerythritol, glutathione, L-cysteine ​​and β-mercaptoethanol; More preferably, step b) includes incubation at a temperature of at least 20°C for at least 30 minutes in the presence of 2-mercaptoethylamine, or in the presence of dithiothreitol, or in the presence of tris(2-carboxyethyl)phosphine or its chemical derivatives thereof.

27. The production method according to claim 26, wherein Step c) or step c-1) includes removing the reducing agent; preferably, step c) or step c-1) further includes natural oxidation or the addition of an oxidizing agent; preferably, step c) or step c-1) is a purification step, removing the homodimer from the reaction system and purifying to obtain the heterodimer polypeptide conjugate or heterodimer polypeptide.

28. A heterodimeric polypeptide conjugate prepared by any one of claims 1-27.

29. A pharmaceutical composition comprising the heterodimeric polypeptide conjugate of claim 28; preferably, further comprising one or more pharmaceutically acceptable carriers.

30. The use of the heterodimeric polypeptide conjugate of claim 28 or the pharmaceutical composition of claim 29 in the preparation of a medicament for treating and / or preventing a disease; preferably, the disease is a tumor.

31. A method for treating and / or preventing a disease in a subject, comprising administering to the subject the heterodimeric polypeptide conjugate of claim 28 or the pharmaceutical composition of claim 29; preferably, the disease is a tumor.

32. The heterodimeric polypeptide conjugate of claim 28 or the pharmaceutical composition of claim 29, for the treatment and / or prevention of a disease, or as a medicament for the treatment and / or prevention of a disease; preferably, the disease is a tumor.

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