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WO2025162460A1 - Composé composé, et préparation et utilisation associées - Google Patents

Composé composé, et préparation et utilisation associées

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Publication number
WO2025162460A1
WO2025162460A1 PCT/CN2025/075554 CN2025075554W WO2025162460A1 WO 2025162460 A1 WO2025162460 A1 WO 2025162460A1 CN 2025075554 W CN2025075554 W CN 2025075554W WO 2025162460 A1 WO2025162460 A1 WO 2025162460A1
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WO
WIPO (PCT)
Prior art keywords
connecting peptide
conjugated compound
amino acid
peptide
cys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/075554
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English (en)
Chinese (zh)
Inventor
王峰
张雨菡
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nantong Yichen Biopharma Co Ltd
Original Assignee
Nantong Yichen Biopharma Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nantong Yichen Biopharma Co Ltd filed Critical Nantong Yichen Biopharma Co Ltd
Publication of WO2025162460A1 publication Critical patent/WO2025162460A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention mainly relates to the field of biopharmaceuticals, in particular to a conjugated compound and its preparation and application.
  • ADCs Antibody-drug conjugates
  • linkers to connect monoclonal antibodies to small molecule drugs. Leveraging the specificity of the monoclonal antibody, they precisely target the drug and minimize its toxic side effects on normal cells.
  • ADCs combine the specificity and stability of antibody drugs with the pharmacodynamic properties of small molecule toxins against tumor cells, making them a hot topic in anti-tumor drug research.
  • Traditional ADCs are constructed by reacting the amino group of an antibody's lysine residue with a succinimidyl ester on the linker, or by reacting the thiol group of a reduced cysteine residue with a maleimide on the linker.
  • An antibody molecule contains 80 to 90 lysine residues, and conjugation can occur at nearly 40 different lysine residues. Breaking cysteine disulfide bonds creates multiple cysteine residues, compromising the integrity of the antibody molecule. Consequently, traditional ADCs are highly heterogeneous mixtures (with varying DARs and conjugation sites), resulting in poor stability and prone to aggregation, which compromises efficacy and therapeutic window. Furthermore, analyzing, identifying, and controlling variability between production batches present significant technical challenges. Site-directed conjugation technology enables the targeted and quantitative conjugation of antibodies to small molecule toxins.
  • the ADCs obtained using this technology exhibit an appropriate drug-to-antibody ratio (DAR), high uniformity, good stability, and excellent batch-to-batch reproducibility. They exhibit enhanced activity and pharmacokinetic properties, making them more suitable for large-scale ADC production.
  • Site-directed conjugation technology typically requires the transformation or modification of the antibody, such as the introduction of a thiol-containing mutant amino acid or a non-natural amino acid with an active functional group, to achieve site-directed drug conjugation.
  • the selection of the introduction site, the type of amino acid to be introduced, and the effect of the introduced amino acid on the antibody's structure and activity pose significant challenges to site-directed conjugation. Utilizing the native sequence of the antibody for site-directed conjugation, while maintaining the stability and activity of the conjugate while achieving a uniform DAR, is particularly important.
  • the main purpose of the present invention is to provide a conjugated compound with stable structure and uniform product, so as to solve the problems of heterogeneous drug-antibody ratio (DAR) and unstable product of ADC drugs in the prior art, which further affect the application and drug development of ADC.
  • DAR drug-antibody ratio
  • the first aspect of the present invention provides a conjugated compound comprising an antibody portion and a heterologous portion, wherein the antibody portion comprises two polypeptide chains:
  • the first polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A1, B1, C1, D1, and E1;
  • the second polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A2, B2, C2, D2, and E2;
  • the C1 binding module is the heavy chain constant region CH1;
  • the C2 binding module is the light chain constant region, CL, and the cysteine 214 at position CL is deleted (C214del, EU numbering, all numbers are used in the EU) or mutated to serine (Ser) (C214S), glycine (Gly) (C214G), or threonine (Thr) (C214T);
  • the D1 binding module is the first Fc
  • the D2 binding module is the second Fc
  • the B1 and/or B2 binding moieties are independently selected from a heavy chain variable region (VH), a light chain variable region (VL), a single domain antibody, a VHH domain, a ligand binding domain of a receptor, a receptor binding domain of a ligand, a non-immunoglobulin antigen binding scaffold;
  • VH heavy chain variable region
  • VL light chain variable region
  • Single domain antibody a VHH domain
  • a ligand binding domain of a receptor a receptor binding domain of a ligand, a non-immunoglobulin antigen binding scaffold
  • the A1 and/or A2 binding moieties are absent or independently selected from a heavy chain variable region (VH), a light chain variable region (VL), a single domain antibody, a VHH domain, a ligand binding domain of a receptor, a receptor binding domain of a ligand, a non-immunoglobulin antigen binding scaffold, a single variable domain of a TCR;
  • the E1 and/or E2 binding moieties are absent or are independently selected from a heavy chain variable region (VH), a light chain variable region (VL), a single domain antibody, a VHH domain, a ligand binding domain of a receptor, a receptor binding domain of a ligand, a non-immunoglobulin antigen binding scaffold;
  • VH heavy chain variable region
  • VL light chain variable region
  • Single domain antibody a VHH domain
  • a ligand binding domain of a receptor a receptor binding domain of a ligand, a non-immunoglobulin antigen binding scaffold
  • the C1 binding module and the D1 binding module are connected by a first connecting peptide, and the C2 binding module and the D2 binding module are connected by a second connecting peptide;
  • the first connecting peptide and the second connecting peptide comprise one or more cysteine (Cys) residues to which the heterologous moiety is conjugated.
  • one of the B1 and B2 binding moieties is selected from VH
  • the other of the B1 and B2 binding moieties is selected from VL
  • VH and VL form a first binding domain
  • one of the A1 and A2 binding moieties is selected from VH2
  • the other of the A1 and A2 binding moieties is selected from VL2
  • VH2 and VL2 form a second binding domain.
  • one of the E1 and E2 binding moieties is selected from VH3, the other of the E1 and E2 binding moieties is selected from VL3, and VH3 and VL3 form a third binding domain.
  • A1, A1, E1, E2 are absent, and one of the B1 and B2 binding moieties is VH, and the other of the B1 and B2 binding moieties is VL, and VH and VL form the first antigen binding domain.
  • E1 and E2 are absent, and one of the B1 and B2 binding moieties is VH, the other of the B1 and B2 binding moieties is VL, one of the A1 and A2 binding moieties is VH2, the other of the A1 and A2 binding moieties is VL2, VH and VL form a first antigen binding domain, and VH2 and VL2 form a second antigen binding domain.
  • A1 and A2 are absent, and one of the B1 and B2 binding moieties is VH, the other of the B1 and B2 binding moieties is VL, one of the E1 and E2 binding moieties is VH3, the other of the E1 and E2 binding moieties is VL3, VH and VL form a first antigen binding domain, and VH3 and VL3 form a third antigen binding domain.
  • A1, A2, B1, B2 are absent, and one of the E1 and E2 binding moieties is VH3, and the other of the E1 and E2 binding moieties is VL3, and VH3 and VL3 form a third antigen binding domain.
  • the antibody in the conjugated compound is a bispecific antibody targeting EGFR and cMet.
  • the first chain of the bispecific antibody targeting EGFR and cMet comprises, from N-terminus to C-terminus, the following sequence: the heavy chain variable region (VH2) of the antibody targeting cMet, a first connecting peptide, the heavy chain variable region (VH1) or light chain variable region (VL1) of the antibody targeting EGFR, CH1, a first hinge region, and a first Fc;
  • the second chain comprises, from N-terminus to C-terminus, the following sequence: the light chain variable region (VL2) of the antibody targeting cMet, a second connecting peptide, the light chain variable region (VL1) or heavy chain variable region (VH1) of the antibody targeting EGFR, CL, a second hinge region, and a second Fc.
  • VH2 has HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 53, SEQ ID NO. 54, and SEQ ID NO. 55, respectively
  • VL2 has LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 56, SEQ ID NO. 57, and SEQ ID NO. 58, respectively.
  • the amino acid sequences of VH2 and VL2 are shown in SEQ ID NO. 38 and SEQ ID NO. 40, respectively.
  • the HCDR1, HCDR2, and HCDR3 of VH1 are selected from the group consisting of SEQ ID NO. 59-61, SEQ ID NO. 65-67, or SEQ ID NO.
  • the LCDR1, LCDR2, and LCDR3 of VL are selected from the group consisting of SEQ ID NO. 62-64, SEQ ID NO. 68-70, or SEQ ID NO. 74-76, respectively.
  • the amino acid sequences of VH1 and VL1 are shown as SEQ ID NO.42 and SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.48, or SEQ ID NO.50 and SEQ ID NO.52, respectively.
  • the two chains of the bispecific antibody targeting EGFR and cMet are as follows: SEQ ID NO.1 and SEQ ID NO.2; SEQ ID NO.3 and SEQ ID NO.4; SEQ ID NO.5 and SEQ ID NO.6; SEQ ID NO.7 and SEQ ID NO.8; SEQ ID NO.9 and SEQ ID NO.10; SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.77 and SEQ ID NO.78, SEQ ID NO.79 and SEQ ID NO.80, SEQ ID NO.81 and SEQ ID NO.82, SEQ ID NO.83 and SEQ ID NO.84, SEQ ID NO.85 and SEQ ID NO.86, SEQ ID NO.87 and SEQ ID NO.88, SEQ ID NO.89 and SEQ ID NO.
  • first connecting peptide and the second connecting peptide are polypeptides with a length of 5-20 amino acids.
  • first connecting peptide and the second connecting peptide may be the same or different.
  • the first connecting peptide and the second connecting peptide are flexible peptides; further, the flexible peptide contains glycine (Gly) and/or serine (Ser); further, the flexible peptide contains glycine (Gly), serine (Ser), threonine (Thr), alanine (Ala), glutamic acid (Glu) and/or phenylalanine (Phe).
  • the flexible peptide contains glycine (Gly) and/or serine (Ser); further, the flexible peptide contains glycine (Gly), serine (Ser), threonine (Thr), alanine (Ala), glutamic acid (Glu) and/or phenylalanine (Phe).
  • first connecting peptide and the second connecting peptide are rigid peptides; further, the rigid peptides are composed of ⁇ -helices; further, the rigid peptides contain glutamic acid (Glu), alanine (Ala) and/or lysine (Lys).
  • the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.137:EPKSCDKTHTCPPCP; further, one or two Cys at positions 220, 226 and 229 of the first connecting peptide and the second connecting peptide on SEQ ID NO.137 are deleted and/or mutated; further, one or two Cys at positions 220, 226 and 229 of the first connecting peptide and the second connecting peptide on SEQ ID NO.137 are mutated to Ser, Thr or Gly; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 166, 167, 168, 169, 170, 171.
  • the first connecting peptide and the second connecting peptide are the hinge of IgG2; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.156:ERKCCVECPPCP(E216-P230); further, the first connecting peptide and the second connecting peptide have one or two Cys deletions and/or mutations at positions 219, 220, 226 and 229 of SEQ ID NO.156; further, the amino acid after the Cys mutation is Ser, Thr or Gly; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.157, 158, 159, 160, 161, 162, 163 or 164.
  • the first connecting peptide and the second connecting peptide are the hinge of IgG4; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.150:ESKYGPPCPPCP(E216-P230); further, one of the Cys at positions 226 and 229 of SEQ ID NO.150 of the first connecting peptide and the second connecting peptide is missing or mutated; further, the amino acid after the Cys mutation is Ser, Gly or Thr; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.151, 152, 154 or 155.
  • first connecting peptide and the second connecting peptide are the hinge of IgG3; further, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.165:ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3.
  • the first Fc and the second Fc comprise modified CH3 domains, wherein the modified CH3 domains comprise amino acid substitutions that promote heterologous pairing between the first Fc and the second Fc; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitution T366W, and the other of the first Fc and the second Fc comprises amino acid substitutions T366S, L368A, and Y407V; in a specific embodiment, one of the first Fc and the second Fc comprising amino acid substitution T366W further comprises amino acid substitution S354C, and the other of the first Fc and the second Fc comprising amino acid substitutions T366S, L368A, and Y407V further comprises one of the amino acid substitutions further comprises Y349C; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions E356K and R409K, and the other of the first Fc and the second Fc comprises amino acid substitutions R409K
  • the heterologous portion of the conjugated compound is a substance suitable for tumor targeting, disease diagnosis, cure, alleviation, treatment or prevention
  • the heterologous portion is a drug, a toxin, a CDK inhibitor, an HDAC inhibitor, a TLR agonist, a PROTAC protein degrader, a radionuclide, an immunomodulator, urea glutamate (DUPA) and its analogs, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a peptide nucleic acid, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, an unnatural amino acid, a peptide, a lipid, a carbohydrate, a scaffold molecule, a fluorescent tag, a visualization
  • the heterologous moiety of the conjugated compound is selected from an ethyleneimine derivative, a triazene, a folic acid analog, an anthracycline, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an epipodophyllotoxin, a platinum coordination compound, a vinca alkaloid, a substituted urea, an adrenocortical suppressant, a hormone antagonist, endostatin, camptothecin, a camptothecin derivative, SN-38, doxorubicin, a doxorubicin analog, an antimetabolite, an alkylating agent, an antimitotic agent, an antiangiogenic agent, an mTOR inhibitor, a heat shock protein inhibitor, a proteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, methotrexate, CPT-11, or a combination thereof.
  • the drug in the conjugate compound is selected from nitrogen mustard, alkyl sulfonate, nitrosourea, gemcitabine, methylhydrazine derivatives, paclitaxel, tyrosine kinase inhibitors, or a combination thereof; in a specific embodiment, the drug is selected from auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Dinaciclib, Mocetinostat, Vorinostat, Olaparib, Ceralasertib, M35 41.
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • Dinaciclib Mocetinostat
  • Vorinostat Vorinostat
  • Olaparib Ceralasertib, M35 41.
  • CC-885 Dx-8951, tubulysin, exatecan derivative (Dxd), pyrrolobenzodiazepine (PBD), maytansinoid, DM1, DM4, calicheamicin, duocarmycin (CAS NO. 130288), duostatin, duostatin-3, duostatin-5, rachelmycin (CC-1065), SN-38, SG3199, or doxorubicin.
  • the heterologous moiety in the conjugated compound is selected from urea glutamate (DUPA).
  • the heterologous moiety in the conjugated compound is conjugated to the Cys of the first connecting peptide and/or the second connecting peptide via a linker.
  • the linker in the conjugated compound is selected from a peptide linker, a non-peptide linker, a cleavable linker or a non-cleavable linker.
  • Another aspect of the present invention provides a method for preparing the aforementioned conjugated compound, which comprises placing the antibody portion under reducing conditions so that the thiol groups of one or more Cys of the first connecting peptide and the second connecting peptide are reduced, and then reacting the reduced thiol groups with an active agent; preferably, the thiol group reacts with the heterologous portion through a Michael reaction using a linker containing a maleimide group.
  • compositions comprising the aforementioned conjugated compound and a pharmaceutically acceptable carrier, diluent or excipient.
  • cancer such as epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, prostate cancer, colon cancer, rectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, or sporadic or hereditary papillary renal cell carcinoma, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrous carcinoma, leukemia or lymphoma, etc.), autoimmune diseases, inflammatory or infectious diseases, etc.
  • cancer such as epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, prostate cancer, colon cancer, rectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, pharyngeal cancer, nasal cancer, pan
  • the present invention discovered that after the disulfide bond of the Cys on the first and second connecting peptides of the antibody portion is opened using the reducing agent Tcep (tris(2-carbonylethyl)phosphine hydrochloride), the heterologous portion is conjugated to the reduced thiol group of Cys.
  • Tcep tris(2-carbonylethyl)phosphine hydrochloride
  • the conjugated compound thus obtained has a stable structure (see the bands between 100-130 kD on the non-reducing SDS-PAGE of Figures 2P and 2Q); in addition, the further added A1 and A2
  • the non-covalent interaction of VH2/VL2 formed by the binding module further enhances the stability of the obtained conjugated compound (see the bands near 130KD in Figures 2F, 2I, 2N, and 2O), while the conjugated compounds obtained by other ADC coupling technologies currently on the market appear as multiple bands on non-reducing SDS (see lanes 2-4 of aEGFR mAb in Figure 2F and lanes 2-3 of aHER2 mAb in Figure 2I).
  • the DAR value of the obtained conjugated compound is more uniform than that of other conjugated compounds (as shown in Figure 4), which is conducive to ensuring the consistency of its clinical efficacy and safety.
  • FIG1 is a schematic diagram of the antibody structure of the conjugated compound.
  • Figures 2A-2Q are SDS-PAGE gel images of antibodies and their conjugated compounds, "-" and “+” represent no reducing agent and reducing agent, respectively;
  • Figure 2F and Figure 2I are sample treatment conditions without reducing agent, "1” in Figure 2F and Figure 2I represents the uncoupled double antibody treated with (tris (2-carbonylethyl) phosphine hydrochloride) (Tcep), “2” represents the antibody coupled with Dxd after Tcep treatment, “3” represents the double antibody coupled with MMAE after Tcep treatment, and “4" represents the double antibody coupled with Dx8951 after Tcep treatment;
  • Figure 2K is an SDS-PAGE gel image with the addition of reducing agent DTT, and lanes 1-10 are MTE-6263, MTE-6465 ...
  • Figure 3A and Figure 3B show the gel exclusion chromatography of the bispecific antibody and its conjugate.
  • Figure 4A- Figure 4Y shows the mass spectrometric identification of the coupling products based on MMAE or Dxd.
  • Figures 5A-5T are the ELISA test results of the antigen binding activity of the bispecific antibodies and their conjugates.
  • 6A-6D are flow cytometry detection results of the binding of bispecific antibodies to cell surface antigens.
  • FIG7A-FIG7B show the endocytosis mediated by bispecific antibodies on different cell lines.
  • Figures 8A-8K show the specific cell-killing activity of MMAE-based bispecific antibody-drug conjugate products.
  • FIG9 shows the specific cell-killing activity of the Dxd-based bispecific antibody-drug conjugate product.
  • FIG10A and FIG10B respectively show the inhibitory effect of BEC-8283 and its MMAE conjugate on tumor and the effect on body weight of NCI-H1975 tumor-bearing mice.
  • FIG11A and FIG11B show the inhibitory effects of BEC-8283 and its Dxd conjugate on tumor and the effects on body weight in HCC827 tumor-bearing mice, respectively.
  • a “conjugated compound” includes an antibody portion and a heterologous portion, wherein the antibody portion comprises two polypeptide chains: the first polypeptide chain comprises, from N-terminus to C-terminus, A1, B1, C1, D1, and E1 binding modules, and the second polypeptide chain comprises, from N-terminus to C-terminus, A2, B2, C2, D2, and E2 binding modules, wherein the C1 binding module is the heavy chain constant region CH1, the C2 binding module is the light chain constant region CL, the D1 binding module is the first Fc, the D2 binding module is the second Fc, the B1 and/or B2 binding modules may be absent or independently selected from, but not limited to, a heavy chain variable region (VH), a light chain variable region (VL), a single domain antibody, a VHH domain, a ligand binding domain of a receptor, a receptor binding domain of a lig
  • the "connecting peptide” of the present invention is mainly used to provide Cys for coupling to the heterologous part.
  • the connecting peptide can contain one, two, three or even more Cys in order to obtain a conjugate of a specific DAR. Therefore, the "connecting peptide” of the present invention can be a flexible peptide, a rigid peptide or a combination of flexible and rigid peptides.
  • the "first connecting peptide” and the “second connecting peptide” only indicate the polypeptide chain in which they are located, rather than limiting their sequences.
  • the “first connecting chain” and the “second connecting peptide” can be the same or different, as long as the Cys thereon can form a correct and stable pairing between the chains.
  • the "connecting peptide” can be a flexible peptide, a rigid peptide or composed of a flexible peptide and a rigid peptide.
  • the connecting peptide of the present invention can be selected from the hinge of IgG1, IgG2, IgG3, IgG4 or a combination thereof.
  • the connecting peptide has any sequence such as SEQ ID NO.137-149, SEQ ID NO.166-171 or a combination thereof.
  • the connecting peptide has any one of SEQ ID NOs. 156-164 or a combination thereof.
  • the connecting peptide has any one of SEQ ID NOs. 150-155 or a combination thereof.
  • the connecting peptide has the sequence shown in SEQ ID NO. 165.
  • the first and second Fc regions of the present invention comprise modified CH3 domains, wherein the modified CH3 domains comprise amino acid mutations that promote heterodimer formation between the first and second Fc regions (e.g., chimeric mutations, complementation mutations, locking and docking mutations, knobs into holes mutations, charge mutations, strand exchange engineered domain (SEED) mutations, etc.). Therefore, the first and second Fc regions can be selected from IgG, IgA, IgE, or IgM isotypes; further, the first and second Fc regions can be independently selected from IgG1, IgG2, IgG3, or IgG4.
  • the modified CH3 domains comprise amino acid mutations that promote heterodimer formation between the first and second Fc regions (e.g., chimeric mutations, complementation mutations, locking and docking mutations, knobs into holes mutations, charge mutations, strand exchange engineered domain (SEED) mutations, etc.). Therefore, the first and second Fc regions can be selected from
  • one of the first Fc and the second Fc comprises the amino acid substitution T366W, and the other of the first Fc and the second Fc comprises the amino acid substitutions T366S, L368A, and Y407V; in a specific embodiment, one of the first Fc and the second Fc comprises the amino acid substitutions T366W and S354C, and the other of the first Fc and the second Fc comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V; in a specific embodiment, one of the first Fc and the second Fc comprises the amino acid substitutions E356K and R409K, and the other of the first Fc and the second Fc comprises amino acid substitutions R409K and K439E; in a specific embodiment, one of the first Fc and the second Fc comprises the amino acid substitution K409R, and the other of the first Fc and the second Fc comprises the amino acid substitution L368E; in a specific embodiment,
  • a "heterologous moiety” is a substance suitable for tumor targeting, disease diagnosis, cure, alleviation, treatment or prevention.
  • the heterologous moiety is selected from drugs, toxins, CDK inhibitors, HDAC inhibitors, TLR agonists, PROTAC protein degraders, radionuclides, immunomodulators, urea glutamate (DUPA) and its analogs, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factor, hormones, hormone antagonists, enzymes, oligonucleotides, DNA, RNA, siRNA, RNAi, microRNA, peptide nucleic acids, photoactive therapeutic agents, anti-angiogenic agents , apoptotic agents, non-natural amino acids, peptides, lipids, carbohydrates, scaffold molecules, fluorescent tags, visualization peptides, biotin, serum half-life regulators, capture tags, chelating agents and solid supports; preferably, the drug is selected from the group consisting
  • the reducing conditions used in the preparation of the conjugated compound of the present invention are provided by a reducing agent.
  • the interchain disulfide bond reducing agent used in the preparation can be any reagent suitable for reducing interchain cysteine disulfide bonds. Suitable interchain disulfide bond reducing agents are well known to technicians, see, for example, R.E.Hansen et al. Analytical Biochemistry, 2009, 394, 147-158. Generally, they are water-soluble and have a negative redox potential at pH 7.
  • the reducing agent can be a thiol or a phosphine.
  • Suitable thiols include 1,4-(dithiobutyl)-2-amine (DTBA), glutathione, cysteine, 2-mercaptoethanol, 2-mercaptoethylamine, dithioerythritol (DTE) or dithiothreitol (DTT).
  • Suitable phosphines include tris(3-sulfophenyl)phosphine, tris(2-hydroxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP) or tris(hydroxymethyl)phosphine.
  • Preferred reducing agents are tris(3-sulfophenyl)phosphine, TCEP and DTT.
  • the most preferred interchain disulfide bond reducing agent is TCEP.
  • VL and VH gene fragments that constitute the antibody and the gene fragment of the antibody constant region were synthesized separately, and the above gene fragments were amplified by PCR.
  • the amplified VH gene fragment was connected to the gene fragment of the antibody constant region by overlap PCR as the first chain; the amplified VL gene fragment was connected to the gene fragment of the antibody constant region as the second chain, as shown in Figure 1; the above fragments were further connected to the pFuse vector used for eukaryotic expression (InvivoGen, CA) through homologous recombination, and transformed into Escherichia coli competent DH5 ⁇ cells.
  • Antibiotic screening was performed on LB plates. After selecting positive clones, plasmids were extracted using an endotoxin-free plasmid extraction kit, and the extracted plasmid sequences were sequenced and verified.
  • the nucleotide and amino acid sequences of each construct are as follows:
  • the eukaryotic expression vector plasmid constructed in Example 1 was co-transfected into FreeStyle HEK293 cells at a cell density of 2.5 x 106 cells/ml and cultured at 125 rpm, 37°C, and 5% CO2 for 5-6 days.
  • the cell culture supernatant was collected by centrifugation and filtered through a 0.22 ⁇ m filter.
  • the fusion antibody was purified using Protein A Resin (Genscript) according to the manufacturer's instructions. The concentration was determined by A280 and BCA assays (Pierce).
  • the fusion antibody purified using Protein A Resin was further isolated, purified, and buffer exchanged using a GE AKTA chromatography system and a Superdex 200 Increase 10/300 GL gel exclusion chromatography column in PBS buffer (pH 7.4).
  • the purified sample was stored in PBS buffer (pH 7.4).
  • the composition and purity of the antibody were determined by SDS-PAGE under reducing and non-reducing conditions.
  • the monomer components of the antibody were analyzed in a saline solution environment by gel exclusion chromatography.
  • the reduction reaction involves reacting the antibody and TCEP at a 1/20 molar ratio in a 37°C incubator for 60 minutes. The sample is then removed and cooled on ice. A pre-chilled payload with a linker is then added at a 20/1 molar ratio to the antibody. The reaction is continued on ice for 2 hours. The TCEP introduced by the reduction reaction and excess unreacted payload are removed using a 40kDa Zeba desalting spin column (CAS: 87766).
  • the coupled sample is then replaced with a suitable buffer system (10 mg/mL sucrose, 20 mg/mL glycine, 1.47 mg/mL glutamic acid, pH 4.0) from the previous DPBS buffer system.
  • a suitable buffer system (10 mg/mL sucrose, 20 mg/mL glycine, 1.47 mg/mL glutamic acid, pH 4.0) from the previous DPBS buffer system.
  • the coupled product is then analyzed for composition and purity by SDS-PAGE under both reducing and non-reducing conditions.
  • the antibody coupled product is then analyzed by gel exclusion chromatography.
  • the conjugated and unconjugated antibodies from Example 2 were mixed at a concentration of 1 mg/ml with freshly prepared thermal shift dye and shift buffer (Protein Thermal Shift TM Dye Kit, ThermoFisher Scientific, Cat. 4461146) in the manufacturer's recommended ratios.
  • Thermal scanning was performed at a heating rate of 0.05°C/s from 25°C to 99°C using a ViiA TM 7 Real-Time PCR System.
  • the melting temperature (Tm) was calculated using the "Area under the curve (AUC)" analysis model in GraphPad Prism 7 software. Atelizumab (Genscript) was used as a monoclonal antibody control. Each data set was replicated twice to ensure reproducibility. The results are shown in Table 2-1.
  • the thermal melting temperature (Tm) of the bispecific antibody is similar to the Tm value of the control antibody Atezolizumab; the Tm value of the product of the bispecific antibody coupled with Dxd or MMAE is close to the Tm of the bispecific antibody, indicating that the bispecific antibody and its coupling product have similar thermal stability to the monoclonal antibody Atezolizumab.
  • the bispecific antibody and conjugated product from Example 2 were incubated with PNGase F (NEB) at a concentration of 1 mg/ml overnight at 37°C.
  • the deglycosylated sample was reduced by adding 10 mM DTT and injected onto a 300SB-C8, 2.1 x 50 mm column on an HPLC-Q-TOF-MS (Agilent, USA). MS was performed, and the DAR value was calculated using the "Aera Under Curve” (AUC) function in GraphPad Prism 7 software.
  • Cell lines with antibody-targeted antigens were cultured in 10% FBS-containing DMEM medium for adherence and then trypsinized. 2x10e5 cells were aliquoted into each flow cytometry well and blocked with pre-chilled 2% FBS-PBS blocking buffer for 30 minutes. The bispecific antibody and corresponding control antibody samples were diluted to 200nM in blocking buffer. Using this as the starting concentration, a 5-fold serial dilution was performed, with a total of 8 dilutions. A 0nM concentration was set as the staining background. 100ul of diluted antibody sample was added to each well and incubated at 4°C for 30 minutes.
  • the cell lines A549 and SKBR3 with large differences in the abundance of dual target antigens in Example 6 were selected.
  • the above cells were digested with trypsin and resuspended in DMEM complete medium containing 10% FBS.
  • 2x10e5 cells were dispensed into each flow staining well and blocked with pre-cooled 2% FBS-PBS blocking solution for 30 minutes.
  • the control antibodies BEC-6162 and BEC-6364 in Example 2 were diluted to 1 ⁇ M, respectively, and added to the above flow staining wells to block the target antigens.
  • the cells were incubated at 4°C for 1 hour, washed 3 times with PBS, and the labeled APC-YF-382+383 sample diluted to 50 nM was added to the washed flow tube and incubated at 4°C for 30 minutes. After washing once with PBS, the above flow staining was added. The tubes were placed in a 37°C incubator to allow for endocytosis. Samples were removed at different time points and washed once with PBS. Each sample was then aliquoted into two aliquots.
  • Example 8 Killing activity of MMAE-coupled products
  • Culture cell lines with antibody-targeted antigens on their surfaces Take a certain amount of the above-mentioned suspended cells, centrifuge at 500g, discard the supernatant, and resuspend the cells in 1640 complete medium containing 10% FBS to a density of 1x10e4 cells/mL. Use an 8-channel pipette to draw 100 ⁇ L into a 96-well clear-bottom black plate (CAS: 060096). Culture the cells in a cell culture incubator with 5% CO2. First, prepare 10 ⁇ EGFR monoclonal antibody STOCK (10 ⁇ M) and cMET monoclonal antibody STOCK (10 ⁇ M) in DPBS buffer.
  • EGFR monoclonal antibody STOCK and cMET monoclonal antibody STOCK were added to the competitive binding group, with a final concentration of 1uM, and pre-incubated with the cells added to the well plate for 5 hours; the bispecific antibody and the corresponding control antibody drug conjugate product were diluted with DPBS buffer to a maximum concentration of 100nM, and then diluted 3-fold in series.
  • the MMAE-based bispecific antibody BEC-8283 conjugate product has a good killing effect on positive cells, especially double-positive cells; when cMET and EGFR were unilaterally blocked, the cytotoxicity of the bispecific antibody conjugate product was reduced, especially after unilateral blocking of EGFR, the cytotoxicity was significantly reduced, suggesting that the EGFR side plays a dominant killing role in the bispecific antibody conjugate product.
  • the positive cell line HCC827 was used to evaluate the target-dependent toxicity mediated by the specific antibody and the corresponding control antibody-drug conjugate product.
  • the cell line with the antibody-targeted antigen on the surface was cultured. A certain amount of the above-mentioned suspended cells was taken, centrifuged at 500g and the supernatant was discarded. The cells were resuspended in 1640 complete medium containing 10% FBS to a density of 2x10e4Cells/mL. 100 ⁇ L was drawn into a 96-well transparent bottom black plate (CAS: 060096) using an 8-channel pipette. The cells were cultured in a cell culture incubator with 5% CO2.
  • the bispecific antibody and the corresponding control antibody-drug conjugate product were diluted with DPBS buffer to a final concentration of 100 nM, and then diluted 3 times in series. After the cells were cultured overnight, they were added and mixed with the cells. The cells were cultured in a cell culture incubator with 5% CO2 for 5 days. After 5 days, the 96-well plate was removed and an equal volume of Cell Titer Glo ( After incubation at room temperature for 15 minutes using the Luminescent Cell Viability Assay (CAS: G7571), the assay was read on a microplate reader. IC50 values were calculated using the "log (inhibitors) vs. response - variable slope (four parameters)" analysis model in GraphPad Prism 7 software. Two replicates were performed for each data set to ensure reproducibility. The results, as shown in Figure 9, demonstrate that the DXD-based bispecific antibody BEC-8283 exhibits a robust cytotoxic effect on double-positive cells.
  • the bispecific antibody was administered to 6-8 week old C57BL6J mice via tail vein.
  • the bispecific antibody was only administered at a high dose of 10 mg/kg.
  • the bispecific antibody conjugate product had three dosing doses, namely 8 mg/kg, 4 mg/kg, and 1 mg/kg. Eye blood was collected at different time points. The concentrations of the intact bispecific antibody and the bispecific antibody conjugate product in the serum samples at each time point were detected by ELISA, and the data were processed by GraphPad Prism.
  • the pharmacokinetic results of the bispecific antibody and its conjugate product in mice are shown in Table 6:
  • Nude mice aged 6-8 weeks were subcutaneously inoculated with tumor cells. 5x10e6 NCI-H1975 or 5x10e6 cells were inoculated on day 0. On day 8, when tumors were approximately 100-200 mm3, a single dose of the bispecific antibody conjugate was administered via the tail vein. Doses of 5, 2.5, and 1 mg/kg of the bispecific antibody were administered, corresponding to doses of 5, 2.5, and 1 mg/kg of the bispecific antibody. Tumor size was monitored three times weekly. As shown in Figure 10, the MMAE bispecific antibody conjugate demonstrated significant NCI-H1975 tumor inhibition.

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Abstract

L'invention concerne un composé conjugué comprenant une fraction d'anticorps et une fraction hétérologue, la fraction d'anticorps contenant une première chaîne polypeptidique comprenant des modules de liaison A1, B1, C1, D1 et E1 séquentiellement de l'extrémité N-terminale à l'extrémité C-terminale, et une seconde chaîne polypeptidique comprenant des modules de liaison A2, B2, C2, D2 et E2 séquentiellement de l'extrémité N-terminale à l'extrémité C-terminale ; et la fraction hétérologue est conjuguée à un résidu de cystéine (Cys) d'une première région charnière située entre les modules C1 et D1 et/ou une seconde région charnière située entre les modules C2 et D2. Un composé conjugué ayant une structure stable et un DAR hautement uniforme est obtenu au moyen d'une interaction non covalente d'A1/A2, B1/B2, C1/C2, D1/D2 et E1/E2. L'invention concerne en outre la préparation du composé conjugué et son utilisation.
PCT/CN2025/075554 2024-02-04 2025-01-27 Composé composé, et préparation et utilisation associées Pending WO2025162460A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018107116A1 (fr) * 2016-12-09 2018-06-14 Abbvie Stemcentrx Llc Procédés de réduction de la toxicité de conjugués anticorps-médicament, et compositions produites par ceux-ci
WO2020135804A1 (fr) * 2018-12-29 2020-07-02 上海一宸医药科技有限公司 Protéine de fusion hétérodimère
WO2020177717A1 (fr) * 2019-03-04 2020-09-10 上海一宸医药科技有限公司 Nouvelle molécule de liaison bispécifique et conjugué médicament associé
WO2022175595A1 (fr) * 2021-02-16 2022-08-25 Glykos Finland Oy Charges utiles de lieurs et leurs conjugués
WO2023056315A1 (fr) * 2021-09-29 2023-04-06 Modex Therapeutics Polypeptides de liaison à l'antigène, complexes polypeptidiques de liaison à l'antigène et leurs méthodes d'utilisation dans le vih

Family Cites Families (6)

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AR056142A1 (es) * 2005-10-21 2007-09-19 Amgen Inc Metodos para generar el anticuerpo igg monovalente
PL2794905T3 (pl) * 2011-12-20 2020-11-02 Medimmune, Llc Zmodyfikowane polipeptydy dla rusztowań przeciwciał dwuswoistych
JP6273205B2 (ja) * 2012-10-05 2018-01-31 協和発酵キリン株式会社 ヘテロダイマータンパク質組成物
KR20250152665A (ko) * 2013-07-31 2025-10-23 암젠 인크 Ｆc-함유 폴리펩타이드의 안정화
IL270596B1 (en) * 2017-05-25 2025-09-01 Bristol Myers Squibb Co Antibodies comprising modified heavy constant region for use in treating cancer
EP4200338A1 (fr) * 2020-08-20 2023-06-28 Amgen Inc. Protéines de liaison à un antigène avec un disulfure non canonique dans la région fab

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018107116A1 (fr) * 2016-12-09 2018-06-14 Abbvie Stemcentrx Llc Procédés de réduction de la toxicité de conjugués anticorps-médicament, et compositions produites par ceux-ci
WO2020135804A1 (fr) * 2018-12-29 2020-07-02 上海一宸医药科技有限公司 Protéine de fusion hétérodimère
WO2020177717A1 (fr) * 2019-03-04 2020-09-10 上海一宸医药科技有限公司 Nouvelle molécule de liaison bispécifique et conjugué médicament associé
WO2022175595A1 (fr) * 2021-02-16 2022-08-25 Glykos Finland Oy Charges utiles de lieurs et leurs conjugués
WO2023056315A1 (fr) * 2021-09-29 2023-04-06 Modex Therapeutics Polypeptides de liaison à l'antigène, complexes polypeptidiques de liaison à l'antigène et leurs méthodes d'utilisation dans le vih

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