WO2025162460A1 - Conjugated compound, and preparation and use thereof - Google Patents

Abstract

A conjugated compound comprising an antibody moiety and a heterologous moiety, wherein the antibody moiety contains a first polypeptide chain comprising binding modules A1, B1, C1, D1 and E1 sequentially from the N-terminus to the C-terminus, and a second polypeptide chain comprising binding modules A2, B2, C2, D2 and E2 sequentially from the N-terminus to the C-terminus; and the heterologous moiety is conjugated to a cysteine residue (Cys) of a first hinge region located between the C1 and D1 modules and/or a second hinge region located between the C2 and D2 modules. The conjugated compound with a stable structure and highly uniform DAR is obtained by means of non-covalent interaction of A1/A2, B1/B2, C1/C2, D1/D2 and E1/E2. In addition, further provided are a preparation of the conjugated compound and the use thereof.

Description

A conjugated compound and its preparation and application Technical Field

The present invention mainly relates to the field of biopharmaceuticals, in particular to a conjugated compound and its preparation and application.

Background Art

Antibody-drug conjugates (ADCs) use 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.

Brief description of the invention

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.

To achieve the above objectives, 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:

a) The first polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A1, B1, C1, D1, and E1;

b) the second polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A2, B2, C2, D2, and E2;

in,

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;

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;

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.

In a specific embodiment, 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, and VH and VL form a first binding domain.

In a specific embodiment, 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, and VH2 and VL2 form a second binding domain.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, the antibody in the conjugated compound is a bispecific antibody targeting EGFR and cMet. In a specific embodiment, 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. In a specific embodiment, VH2 has HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 53, SEQ ID NO. 54, and SEQ ID NO. 55, respectively, and VL2 has LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO. 56, SEQ ID NO. 57, and SEQ ID NO. 58, respectively. In a specific embodiment, the amino acid sequences of VH2 and VL2 are shown in SEQ ID NO. 38 and SEQ ID NO. 40, respectively. In a specific embodiment, 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. 71-73, respectively, and 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. In a specific embodiment, 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. In a specific embodiment, 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. O.90, SEQ ID NO.91 and SEQ ID NO.92, SEQ ID NO.93 and SEQ ID NO.94, SEQ ID NO.95 and SEQ ID NO.96, SEQ ID NO.117 and SEQ ID NO.118, SEQ ID NO.119 and SEQ ID NO.120, SEQ ID NO.121 and SEQ ID NO.121, SEQ ID NO.123 and SE As shown in Q ID NO.124, SEQ ID NO.125 and SEQ ID NO.126, SEQ ID NO.127 and SEQ ID NO.128, SEQ ID NO.129 and SEQ ID NO.130, SEQ ID NO.131 and SEQ ID NO.132, SEQ ID NO.133 and SEQ ID NO.134, or SEQ ID NO.135 and SEQ ID NO.136.

In a specific embodiment, the first connecting peptide and the second connecting peptide are polypeptides with a length of 5-20 amino acids.

In a specific embodiment, the first connecting peptide and the second connecting peptide may be the same or different.

In a specific embodiment, 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).

In a specific embodiment, the first connecting peptide and the second connecting peptide are rigid peptides; further, the rigid peptides are composed of α-helices; further, the rigid peptides contain glutamic acid (Glu), alanine (Ala) and/or lysine (Lys).

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In a specific embodiment, the 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.

In a specific embodiment, 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 and K439E.

In a specific embodiment, the heterologous portion of the conjugated compound is a substance suitable for tumor targeting, disease diagnosis, cure, alleviation, treatment or prevention, preferably, 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 peptide, biotin, a serum half-life regulator, a capture tag, a chelating agent or a combination thereof.

In a specific embodiment, 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.

In a specific embodiment, 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. 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.

In a specific embodiment, the heterologous moiety in the conjugated compound is selected from urea glutamate (DUPA).

In a specific embodiment, 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.

In a specific embodiment, the linker in the conjugated compound is selected from a peptide linker, a non-peptide linker, a cleavable linker or a non-cleavable linker.

In a specific embodiment, the linker in the conjugated compound is selected from mc (maleimidocaproyl), val-cit (valine-citrulline), mc-val-cit (maleimidocaproyl-valine-citrulline), mc-val-cit-PABC (maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate), mcGGFG, mc-(PEG)8-VA-PABC, MCC (N-maleimidomethyl) Cyclohexane-1-carboxylic acid succinimidyl ester, N-maleimidomethyl cyclohexane-1-carboxylate), Mal-PEG2C2(maleimido-[CH2CH20]2CH2CH2C(＝0)), Mal-PEG3C2(maleimido-[CH2CH20]3CH2CH2C(＝0)) and Mal-PEG6C2(maleimido-[CH2CH20]6CH2CH2C(＝0)).

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.

Another aspect of the present invention provides a composition comprising the aforementioned conjugated compound and a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect of the present invention provides the use of the aforementioned composition in the preparation of a medicament for preventing or treating a disease; further, the diseases to be prevented and treated include but are not limited to 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.

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. By virtue of the non-covalent interaction between the VH/VL formed by the B1 and B2 binding modules, the non-covalent interaction between the C1 binding module (CH1) and the C2 binding module (CL), and the heterologous pairing interaction between the D1 binding module (first Fc) and the D2 binding module (second Fc), 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). In addition, since the Cys involved in the conjugation in the present invention are all on the connecting peptide and have similar reactivity, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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 ... TE-6667, MTE-6869, MTE-7071, MTE-7273, MTE-7475, MTE-7677, MTE-7879, MTE-8081; (2L) is the SDS-PAGE gel image with the addition of reducing agent DTT, lanes 1-11 are BEC-2728, BEC-8283, BEC-2930, BEC-3132, BEC-3334, BEC-3536, BEC-3738, BEC-3940, BEC-4142, BEC-4344, BEC-4546; Figure 2M is the SDS-PAGE gel image with the addition of reducing agent DTT, lanes 1-10 are BEC-92 93, BEC-9495, BEC-9697, BEC-9899, BEC-0001, BEC-0203, BEC-0405, BEC-0607, BEC-0809, BEC-1011; Figure 2N shows BEC-9293 and its conjugates, wherein 1 is BEC-9293, 2 is BEC-9293 treated with Tcep, and 3 is the conjugate of BEC-9293 treated with Tcep coupled with DUPA; Figure 2O shows BEC-9495 and its conjugates, wherein 1 is BEC-9495, 2 is BEC-9495 treated with Tcep, and 3 is BEC-9495 treated with Tcep coupled with DUPA molecule; Figure 2P shows MTE-6465 and its conjugates, wherein 1 is MTE-6465, 2 is MTE-6465 treated with Tcep, and 3 is the conjugate after Tcep treatment and coupling with DUPA molecule; Figure 2Q shows MTE-9091 and its conjugates, wherein 1 is MTE-9091, 2 is MTE-9091 treated with Tcep, and 3 is the conjugate after Tcep treatment with MTE-9091 coupled with DUPA; Figure 2R shows MTE-8687 and its conjugates, wherein 1 is MTE-8687, 2 is MTE-8687 treated with Tcep, and 3 is the conjugate after Tcep treatment with MTE-8687 coupled with DUPA.

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.

Detailed description of the invention

The present invention is described in detail herein by reference using the following definitions and examples.The contents of all patents and publications mentioned herein, including all sequences disclosed in such patents and publications, are expressly incorporated herein by reference.

As used herein, "coupled" and "conjugated" have the same meaning and can be used interchangeably herein. 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 ligand, a non-immunoglobulin antigen binding scaffold, or a single variable domain of a TCR, and the A1 and/or A2 binding modules may be absent or The E1 and/or E2 binding moieties are 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 ligand, a non-immunoglobulin antigen-binding scaffold, or a single variable domain of a TCR; the E1 and/or E2 binding moieties 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 ligand, a non-immunoglobulin antigen-binding scaffold, or a single variable domain of a TCR; the C1 binding moiety and the D1 binding moiety are connected by a first connecting peptide, and the C2 binding moiety and the D2 binding moiety are connected by a second connecting peptide, and the first connecting peptide and the second connecting peptide contain one or more cysteine residues (Cys), and the heterologous part is conjugated to the Cys.

As used herein, the "connecting peptide" of the present invention is mainly used to provide Cys for coupling to the heterologous part. As needed, 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. For example, the "connecting peptide" can be a flexible peptide, a rigid peptide or composed of a flexible peptide and a rigid peptide. Preferably, the connecting peptide of the present invention can be selected from the hinge of IgG1, IgG2, IgG3, IgG4 or a combination thereof. When selected from the hinge region from IgG1, the connecting peptide has any sequence such as SEQ ID NO.137-149, SEQ ID NO.166-171 or a combination thereof. When the hinge is selected from IgG2, the connecting peptide has any one of SEQ ID NOs. 156-164 or a combination thereof. When the hinge is selected from IgG4, the connecting peptide has any one of SEQ ID NOs. 150-155 or a combination thereof. When the hinge is selected from IgG3, the connecting peptide has the sequence shown in SEQ ID NO. 165.

Due to the homodimeric nature of molecules containing Fc regions, 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. In a specific embodiment, 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, one of the first Fc and the second Fc comprises the amino acid substitution F405L, and the other of the first Fc and the second Fc comprises the amino acid substitution K409R; in a specific embodiment, one of the first Fc and the second Fc comprises the amino acid substitutions S364H, F405A, and the other of the first Fc and the second Fc comprises the amino acid substitutions Y349T, T394F; In a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions T350V, L351Y, F405A, and Y407V, and the other of the first Fc and the second Fc comprises amino acid substitutions T350V, T366L, K392L, and T394W; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions K409D and K392D, and the other of the first Fc and the second Fc comprises amino acid substitutions D399K and E356K; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions K360E and K409W, and the first Fc and the other of the second Fc comprises amino acid substitutions Q347R, D399V and F405T; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions F405L, D356E and L358M, and the other of the first Fc and the second Fc comprises amino acid substitutions K409R, D356E and L358M; in a specific embodiment, one of the first Fc and the second Fc comprises amino acid substitutions T366S, L368A, Y407V, D356E and L358M, and the other of the first Fc and the second Fc comprises amino acid substitutions T366W, D356E and L358M.

As used in the present invention, a "heterologous moiety" is a substance suitable for tumor targeting, disease diagnosis, cure, alleviation, treatment or prevention. Preferably, 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 of ethyleneimine derivatives, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxin, platinum coordination compounds, vinca alkaloids, substituted ureas, adrenocortical inhibitors, hormone antagonists, endostatin, camptothecin, camptothecin derivatives, SN-38, doxorubicin, doxorubicin analogs, antimetabolites Preferably, the drug is selected from nitrogen mustard, alkyl sulfonate, nitrosourea, gemcitabine, methylhydrazine derivative, paclitaxel, tyrosine kinase inhibitor, or a combination thereof; More preferably, the drug is selected from auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Dinaciclib, Mocetinostat, Vorinostat, t), Olaparib, Ceralasertib, M3541, CC-885, Dx-8951, tubulysin, exatecan derivative (Dxd, exatecan derivative) pyrrolobenzodiazepine (PBD), maytansinoid, DM1, DM4, calicheamicin, duocarmycin (CAS NO. 130288), duostatin, duostatin-3, duostatin-5, rachelmycin (CC-1065), SN-38, SG3199 or doxorubicin.

The "linker" of the present invention can be selected from a cleavable linker, a non-cleavable linker, a peptide linker or a non-peptide linker; non-limiting examples of the "linker" of the present invention include mc (maleimidocaproyl), val-cit (valine-citrulline), mc-val-cit (maleimidocaproyl-valine-citrulline), mc-val-cit-PABC (maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate), mcGGFG, mc-(PEG)8-VA-PABC, MCC (N-maleimidomethyl) cyclohexane-1-carboxylate), Mal-PEG2C2 (maleimido-[CH2CH20]2CH2CH2C(＝0)), Mal-PEG3C2 (maleimido-[CH2CH20]3CH2CH2C(＝0)) or Mal-PEG6C2 (maleimido-[CH2CH20]6CH2CH2C(＝0)).

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.

Example

The examples are for illustrative purposes only and are not intended to limit the present invention in any way.

Example 1 Vector Construction

The 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 DH5α 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:

Table 1 Amino acid sequence listing

Example 2 Preparation of Antibodies and Their Conjugates

2.1 Antibody Expression and Purification

The eukaryotic expression vector plasmid constructed in Example 1 (see Table 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 μm 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.

2.2 Preparation of Antibody Conjugated Products

In this study, we selected compounds based on two toxin molecules, MMAE and Dxd, with maleimide linkers to prepare conjugated products. A certain amount of the antibody described in Example 2.1 was subjected to an optimized reduction reaction with tris(2-carbonylethyl)phosphine hydrochloride (TCEP, CAS: 51805-45-9) to open the interchain disulfide bonds between the first and second chains of the antibody. The maleimide-linked payload (Deruxtecan (Dxd), CAS: 1599440-13-7; vc-MMAE, CAS: 646502-53-6) was then added. Alkylation occurred via nucleophilic Michael addition of the thiol group, forming a stable thioether bond and producing a relatively uniform conjugated product. 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. 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 results, as shown in Figures 2 and 3, show that the bispecific antibody and its conjugated products exhibit little or no free first or second chains under non-reducing SDS conditions, suggesting that the payload-conjugated products have similar structural stability to the unconjugated antibodies. In contrast, significant free light and heavy chains were observed after the hinge disulfide bond-conjugated payloads on the control monoclonal antibodies aEGFR mAb and aHER2 mAb were observed under non-reducing SDS conditions.

Example 3 Thermodynamic stability test

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 ^™ 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 ^™ 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.

Table 2-1 Tm values of antibodies and their conjugates

Table 2-2 Antibody Tm and Tagg values

Table 2-3 Antibody Tm

Table 2-4 Fusion Antibody Tm

Example 4 Mass Spectrometry Analysis

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. The results are shown in Figure 4: The molecular weights of the bispecific antibody drug and its conjugated product were essentially consistent with the theoretical predictions; both chains of the bispecific antibody conjugated with Dxd or MMAE carried a specific number of small molecule drugs, which was the same as the number of reduced Cys in the linker peptide, and the DAR value was a fixed value.

Example 5 Antigen Binding Activity Detection

Coat the target antigen (100 ng/100 ul/well) on a 96-well ELISA plate and incubate overnight at 4°C. Block with PBST containing 3% skim milk powder (0.5% Tween-20 in PBS) at room temperature for 1 hour, then wash with PBST. Dilute the bispecific antibody and the corresponding coupled product samples to 50 nM with blocking solution, and use this as the starting concentration for a 5-fold gradient dilution, with a total of 8 gradient dilutions. Add 100 ul of diluted bispecific antibody and corresponding control antibody samples to each well and incubate at room temperature for 1 hour. After washing 3 times with PBST, add 100 ul of HRP-labeled goat anti-human Fc monoclonal antibody diluted 1:500 according to the instructions to each well, incubate at room temperature for 1 hour, and then wash 5 times with PBST. After washing, add 100 μl of freshly prepared TMB colorimetric reagent (BioLegend, Cat. 421101) to each well and incubate at room temperature in the dark for 10 min. Then, add 100 μl of 1 M H₂SO₄ to each well and incubate for 2 min for color development. Read the sample at 450 nm on a microplate reader. EC₅₀ values were calculated using the "log (agonist) 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 are shown in Figure 5: The binding activity of the bispecific antibody conjugated products based on Dxd or MMAE to two target antigens was not significantly different from that of the unconjugated bispecific antibody, suggesting that the conjugation of Dxd or MMAE to the Cys on the first and second linker peptides of the antibody does not affect the binding activity of the antibody part to the antigen.

Example 6 Cell surface receptor binding activity detection

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. After washing three times with PBS, 100ul of APC-conjugated anti-human Fc monoclonal antibody diluted according to the instructions was added to each well. The cells were incubated at 4°C for 30 minutes and then washed three times with PBS. 200ul of blocking buffer was added to the washed flow cytometry tube, and the resuspended cell suspension was used for flow cytometry analysis. EC50 values were calculated using the "log (agonist) vs. response - variable slope (four parameters)" analysis model in GraphPad Prism 7 software. Each data set was replicated twice to ensure reproducibility. The results, shown in Table 3 and Figure 6, demonstrate synergistic binding between the EGFR and cMet bispecific antibodies. The ratio of the abundance of the two target antigens on each cell surface is shown in Table 4.

Table 3 EC50 of bispecific antibodies binding to cell surface antigens

Table 4 Detection of the abundance of cell surface receptors for dual target antigens

Example 7 Bispecific Antibody-Mediated Endocytosis

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 μM, 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. One aliquot was incubated with acidic buffer (0.2 M Glycine, 0.15 M NaCl, pH 2.0) at 4°C for 10 minutes, while the other aliquot was blocked with 2% FBS-PBS and incubated at 4°C for the same period. After washing once with PBS, 30 μL of 4% paraformaldehyde fixative was added to the flow cytometer tubes and incubated at room temperature for 20 minutes. After washing once with PBS, 200 μL of blocking buffer was added to the washed flow cytometer tubes, and the resuspended cell suspension was used for flow cytometric analysis. Endocytosis results were analyzed using the "XY Table" tool in GraphPad Prism 7 software. Two replicates were performed for each data set to ensure reproducibility. The results are shown in Figure 7: In A549 cells, after blocking the CMT and EGFR target antigens respectively, APC-BEC-8283-mediated endocytosis showed a difference relative to the non-blocking group at 2 hours, and the difference was the largest at 4 hours; after blocking both CMT and EGFR antigens, APC-BEC-8283-mediated endocytosis was significantly reduced, and the endocytosis rate was lower than that of the single target antigen blocking group; similar results were observed on SKBR3, suggesting that the anti-CMET antibody on the outside and the anti-EGFR antibody on the inside of the bispecific antibody can both produce good endocytosis.

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 μL 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 μM) and cMET monoclonal antibody STOCK (10 μM) in DPBS buffer. μM), 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. After 5 hours, the cells were added and mixed with the cells, and 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 ( Luminescent Cell Viability Assay, CAS: G7571) was incubated at room temperature for 15 minutes and then read on a microplate reader. BAB05 antibody conjugate product (double negative control antibody) was used as a control sample. The IC50 value was calculated using the "log (Inhibitors) vs. response--Variable slope (four parameters)" analysis model of GraphPadPrism7 software. Each set of data was repeated twice to ensure the reproducibility of the results. The results are shown in Figure 8 and Table 5. 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.

Table 5 Specific cell killing by MMAE-based bispecific antibody conjugates

Example 9: Killing activity of DXD-coupled products

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 μL 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.

Example 10 Pharmacokinetic Evaluation in Mice

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: The bispecific antibody conjugate product has a pharmacokinetic performance similar to that of the bispecific antibody backbone, indicating that the stability and integrity of the antibody portion of the present invention are not affected after site-specific conjugation to the DAR=6 small molecule by cysteine, and it still has a good blood drug half-life.

Table 6 PK parameters of bispecific antibodies and their conjugates

Example 11 Pharmacodynamic Evaluation in Mice

11.1 Inhibitory Evaluation in NCI-H1975 Tumor-Bearing Mice

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.

11.2 Inhibitory Evaluation in HCC-827 Tumor-Bearing Mice

Nude mice aged 6-8 weeks were subcutaneously inoculated with 5x106 HCC-827 cells on day 0. A single dose was administered via the tail vein on day 5, when tumors reached approximately 200-500 mm3. Tumor size was monitored three times weekly. As shown in Figure 11, the DXD bispecific antibody conjugate demonstrated significant inhibition of HCC-827 tumors.

Claims (31)

A conjugated compound comprising an antibody portion and a heterologous portion, wherein the antibody portion comprises two polypeptide chains: a) The first polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A1, B1, C1, D1, and E1; b) the second polypeptide chain comprises, from N-terminus to C-terminus, the following binding modules: A2, B2, C2, D2, and E2; in, 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 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, or a single variable domain of a TCR; 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, or 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, or a single variable domain of a TCR; The C1 binding module and the D1 binding module are connected via a first connecting peptide, and the C2 binding module and the D2 binding module are connected via a second connecting peptide; The first connecting peptide and the second connecting peptide comprise one or more cysteine (Cys), and the heterologous moiety is conjugated to the Cys of the first connecting peptide and/or the second connecting peptide; The last cysteine (Cys) in the light chain constant region CL (position 214, EU numbering, EU numbering is used hereinafter) is deleted (C214del). The conjugated compound according to claim 1, wherein the first connecting peptide and the second connecting peptide are polypeptides with a length of 5-20 amino acids. The conjugated compound according to claim 2, wherein the first connecting peptide and the second connecting peptide are flexible peptides or rigid peptides. The conjugated compound according to claim 3, wherein the flexible peptide consists of Cys and glycine (Gly), consists of Cys and serine (Ser), or consists of Cys, Ser and Gly. The conjugated compound according to claim 3, wherein the rigid peptide is composed of an α-helix; preferably, the rigid peptide is composed of Cys, glutamic acid (Glu), alanine (Ala) and/or lysine (Lys). The conjugated compound as described in claim 2, wherein the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.137:EPKSCDKTHTCPPCP(E216-P230). The conjugated compound according to claim 2, wherein one or two Cys at positions 220, 226, and 229 of the first connecting peptide and the second connecting peptide are deleted and/or mutated; Preferably, the amino acid after the Cys mutation is Ser, Gly or Thr. The conjugated compound as described in claim 7, wherein the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.138, 139 or 140. The conjugated compound of claim 2, wherein the first connecting peptide and the second connecting peptide have the amino acid sequence shown in SEQ ID NO. 156:ERKCCVECPPCP(E216-P230) (hinge20 IgG2 WT). The conjugated compound as described in claim 2, wherein one or two Cys at positions 219, 220, 226 and 229 of the first connecting peptide and the second connecting peptide are missing and/or mutated; preferably, the amino acid after the Cys mutation is Ser, Gly or Thr. The conjugated compound as described in claim 10, wherein 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 conjugated compound as described in claim 2, wherein the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in SEQ ID NO.150:ESKYGPPCPPCP(E216-P230). The conjugated compound as described in claim 2, wherein one of the Cys at positions 226 and 229 of the first connecting peptide and the second connecting peptide is missing or mutated; preferably, the amino acid after the Cys mutation is Ser, Gly or Thr. The conjugated compound as described in claim 13, wherein 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. The conjugated compound of any one of claims 1 to 14, wherein 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. The conjugated compound of claim 15, wherein 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. The conjugated compound of claim 16, wherein one of the first Fc and the second Fc comprising the amino acid substitution T366W further comprises the amino acid substitution S354C, and the other of the first Fc and the second Fc comprising the amino acid substitutions T366S, L368A and Y407V further comprises one of the amino acid substitutions further comprises Y349C. The conjugated compound of claim 15, wherein 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 and K439E. The conjugated compound of any one of claims 1 to 18, wherein the heterologous moiety is a substance suitable for disease diagnosis, cure, alleviation, treatment or prevention, or a substance suitable for tumor targeting, preferably, the heterologous moiety 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 peptide, biotin, a serum half-life regulator, a capture tag, a chelator, or a combination thereof. The conjugated compound of claim 19, wherein the heterologous moiety is 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 conjugated compound of claim 19, wherein the heterologous moiety is a nitrogen mustard, an alkyl sulfonate, a nitrosourea, gemcitabine, a methylhydrazine derivative, paclitaxel, a tyrosine kinase inhibitor, or a combination thereof. The conjugated compound of claim 19, wherein the drug is auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Dinaciclib, Mocetinostat, Vorinostat, Olaparib, Ceralasertib, M3541, 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 conjugated compound of claim 19, wherein the heterologous moiety is urea glutamate (DUPA). The conjugated compound according to any one of claims 1 to 23, wherein the heterologous moiety is conjugated to the Cys of the first connecting peptide and/or the second connecting peptide via a linker. The conjugated compound of claim 24, wherein the linker is a peptide linker, a non-peptide linker, a cleavable linker or a non-cleavable linker. The conjugated compound of claim 25, wherein the linker is selected from mc (maleimidocaproyl), val-cit (valine-citrulline), mc-val-cit (maleimidocaproyl-valine-citrulline), mc-val-cit-PABC (maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate), mcGGFG, mc-(PEG)8-VA-PABC, MCC (N-maleimidocaproylmethyl) )cyclohexane-1-carboxylic acid succinimidyl ester, N-maleimidomethyl cyclohexane-1-carboxylate), Mal-PEG2C2(maleimido-[CH2CH20]2CH2CH2C(＝0)), Mal-PEG3C2(maleimido-[CH2CH20]3CH2CH2C(＝0)) and Mal-PEG6C2(maleimido-[CH2CH20]6CH2CH2C(＝0)). A method for preparing a conjugated compound according to any one of claims 1 to 26, comprising subjecting the antibody portion according to any one of claims 1 to 26 to reducing conditions such 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 a heterologous moiety. The method of claim 27, wherein the thiol group is reacted with the heterologous moiety via a Michael reaction using a linker comprising a maleimide group. A composition comprising the conjugated compound according to any one of claims 1 to 26, and a pharmaceutically acceptable carrier, diluent or excipient. Use of the composition as claimed in claim 29 in the preparation of a medicament for preventing or treating a disease. The use according to claim 30, wherein the disease to be prevented or treated is selected from: cancer (such as breast cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, bladder cancer, kidney cancer, liver cancer, thyroid cancer, endometrial cancer, muscle cancer, bone cancer, mesothelioma, vascular cancer, fibrous carcinoma, leukemia or lymphoma, etc.), autoimmune diseases, inflammatory or infectious diseases, etc.