Fusion antibody, and preparation and use thereof

Abstract

A fusion antibody, which comprises a first polypeptide chain consisting of VH, CH1, hinge and a first Fc, and a second polypeptide chain consisting of VL, CL, hinge and a second Fc, wherein one or more cysteine residues (Cys) on the hinge of the first polypeptide chain and the second polypeptide chain are deleted or mutated to other amino acids, and the cysteine residue at position 214 on the CL of the second polypeptide chain is deleted (C214del).

Description

Fusion antibodies and their preparation and use Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a fusion antibody and its preparation and application. Background Art

[0002] Disulfide bonds are crucial for maintaining the structure and function of antibodies and protein drugs, so accurate analysis of their disulfide bonds is essential during the production of antibodies and recombinant protein drugs. For antibodies, mismatched disulfide bonds can affect protein structure and function, and even cause antibody aggregation. Disulfide bond mismatches can occur at any stage of the cell fermentation or purification process, so accurate analysis of disulfide bonds in antibodies and proteins helps researchers promptly identify process problems and adjust process parameters to ensure final product quality.

[0003] Under alkaline conditions, disulfide bonds can be degraded by a β-elimination mechanism to form dehydroalanine and persulfide, which can be further reduced back to cysteine ​​residues. The disulfide bonds between the light and heavy chains in IgG molecules are degraded by a β-elimination mechanism, and the resulting cysteine ​​and dehydroalanine cross-links, resulting in the formation of non-reducible thioether bonds. Such thioether bonds account for approximately 0.4% of recombinant monoclonal IgG1 stored at 4°C and as high as 13.6% in heat-stressed samples (MAbs. 2012 Jan 1; 4(1): 17-23).

[0004] Thioether bond formation can be observed in both in vitro storage and in vivo treatment. Following intravenous administration of IgG1k therapeutic antibodies, the conversion rate of thioether bonds is approximately 0.1% / day, similar to the in vitro conversion rate of 0.09% / day. IgG1λ antibodies have a higher conversion rate than IgG1κ, at 0.16% / day. In therapeutic or natural IgG1 antibodies, thioether bond formation is primarily located at C214 (EU numbering) of the LC and C220 (EU numbering) of the HC (J Biol Chem. 2013 Jun 7; 288(23): 16371-16382).

[0005] In addition, the study further found that the formation of thioether products can be seen in the products expressed and purified in mammalian cells through artificially designed and prepared bispecific antibodies or antibody fusion proteins. The removal of thioether products during large-scale production requires the additional development of corresponding separation and purification methods, which further increases production costs.

[0006] Therefore, developing a method to eliminate the formation of thioether bonds in bispecific antibodies or antibody fusion proteins (thus avoiding immunogenicity) while retaining the stability and effectiveness of bispecific antibodies or antibody fusion proteins is of great significance for the production, storage or in vivo treatment of such drugs.

[0007] Brief description of the invention

[0008] To address the quality inhomogeneities between batches of antibody fusion proteins caused by thioether bond formation during production and storage, as well as the effectiveness and safety risks (such as the greater immunogenic potential brought about by thioether bond formation) during in vivo treatment, the present invention provides a fusion antibody that modifies the cysteine ​​residue Cys at specific sites on the hinge of the two polypeptide chains constituting the fusion antibody, and no interchain thioether product is formed after expression.

[0009] To achieve the above objectives, the first aspect of the present invention provides a fusion antibody comprising:

[0010] (a) a first polypeptide chain comprising an antibody heavy chain variable region (VH), an antibody heavy chain first constant region (CH1), a hinge region, and a first Fc region, connected sequentially from the N-terminus to the C-terminus;

[0011] (b) a second polypeptide chain comprising an antibody light chain variable region (VL), an antibody light chain constant region (CL), a hinge region, and a second Fc, connected sequentially from N-terminus to C-terminus; or

[0012] (a) a first polypeptide chain comprising a VL, a CH1, a hinge, and a first Fc linked sequentially from the N-terminus to the C-terminus;

[0013] (b) a second polypeptide chain comprising a VH, a CL, a hinge, and a second Fc linked sequentially from the N-terminus to the C-terminus;

[0014] Among them, VH and VL constitute the first antigen-binding domain, and the cysteine ​​(Cys) at position 214 of the second polypeptide chain CL is deleted (C214del, EU numbering, all EU numbering is used below), or mutated to serine (Ser) (C214S), glycine (Gly) (C214G) or threonine (Thr) (C214T).

[0015] In some embodiments, the hinges of the first polypeptide chain and the second polypeptide chain are different.

[0016] In some embodiments, the hinge between the first polypeptide chain and the second polypeptide chain does not contain Cys at position 220, ie, C220del.

[0017] In some embodiments, the first polypeptide chain has a hinge as set forth in EPKSDKTHTCPPCP (SEQ ID NO: 82) and the second polypeptide chain has a hinge as set forth in DKTHTCPPCP (SEQ ID NO: 101).

[0018] In some embodiments, the hinges of the first polypeptide chain and the second polypeptide chain are the same.

[0019] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain is derived from IgG1, IgG2, IgG3, or IgG4.

[0020] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain is derived from IgG1.

[0021] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is EPKSCDKTHTCPPCP(E216-P230) (SEQ ID NO: 81).

[0022] In some embodiments, one or more Cys residues on the hinge of the first polypeptide chain and the second polypeptide chain are deleted or mutated to other amino acids.

[0023] In some embodiments, the first Cys on the hinge of the first and second polypeptide chains derived from IgG1 is deleted (C220del).

[0024] In some embodiments, the first Cys on the hinge of the first and second polypeptide chains derived from IgG1 is mutated to other amino acids; preferably, the first Cys is mutated to Ser (C220S), Gly (C220G) or Thr (C220T).

[0025] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is deleted (C220del), the second Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C226del) or mutated to Ser (C226S), Gly (C226G) or Thr (C226T).

[0026] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is mutated to Ser (C220S), Gly (C220G) or Thr (C220T), the second Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C226del) or mutated to Ser (C226S), Gly (C226G) or Thr (C226T).

[0027] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is deleted (C220del), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0028] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is mutated to Ser (C220S), Gly (C220G) or Thr (C220T), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0029] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is deleted (C220del) and the second Cys is deleted (C226del), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0030] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is mutated to Ser (C220S), Gly (C220G) or Thr (C220T), and the second Cys is deleted (C226del), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0031] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is deleted (C220del) and the second Cys is mutated to Ser (C229S), Gly (C229G) or Thr (C229T), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0032] In some embodiments, when the first Cys on the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG1 is mutated to Ser (C220S), Gly (C220G) or Thr (C220T), and the second Cys is mutated to Ser (C226S), Gly (C226G) or Thr (C226T), the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S), Gly (C229G) or Thr (C229T).

[0033] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain is derived from IgG4.

[0034] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG4 is ESKYGPPCPSCP(E216-P230) (SEQ ID NO: 94).

[0035] In some embodiments, Cys at position 200 on CH1 of the first polypeptide chain is deleted (C200del).

[0036] In some embodiments, the Cys at position 200 on the first polypeptide chain CH1 is mutated to other amino acids not containing a sulfhydryl group; preferably, the mutation is to Ser (C200S), Gly (C200G) or Thr (C200T).

[0037] In some embodiments, the hinge of the first polypeptide chain and the second polypeptide chain derived from IgG2 has an amino acid sequence as shown in ERKCCVECPPCP(E216-P230) (SEQ ID NO: 100).

[0038] In some embodiments, the first Fc and the second Fc are derived from IgG1, IgG2, IgG3, or IgG4.

[0039] In some embodiments, the first Fc and the second Fc comprise modified CH3 domains comprising amino acid substitutions that promote heterologous pairing between the first Fc and the second Fc.

[0040] In some embodiments, 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; preferably, 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 that one of the amino acid substitutions further comprises Y349C.

[0041] In some embodiments, 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.

[0042] In some embodiments, one of the N-terminus of the first polypeptide chain and the N-terminus of the second polypeptide chain further comprises VH2, and the other of the N-terminus of the first polypeptide chain and the N-terminus of the second polypeptide chain further comprises VL2, and VH2 and VL2 constitute a second antigen-binding domain.

[0043] In some embodiments, the first binding domain of the fusion antibody binds to the EGFR antigen; in some embodiments, the fusion antibody further comprises a second binding domain that binds to the cMET antigen.

[0044] In some embodiments, the fusion antibody of the present invention has a first polypeptide chain and a second polypeptide chain consisting of the following amino acid sequences: 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: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 33 and SEQ ID NO: 34 SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:43 and SEQ ID NO:44; SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:47 and SEQ ID SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:51 and SEQ ID NO:52; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:55 and SEQ ID NO:56; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:59 and SEQ ID NO:60; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:63 and SEQ ID NO:64; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:67 and SEQ ID NO:68; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:71 and SEQ ID NO:72; SEQ ID NO:77 and SEQ ID NO:78;SEQ ID NO: 79 and SEQ ID NO: 80; SEQ ID NO. 102 and SEQ ID NO: 103; SEQ ID NO: 104 and SEQ ID NO: 105; SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 109 and SEQ ID NO: 110; SEQ ID NO: 111 and SEQ ID NO: 112.

[0045] The second aspect of the present invention provides a fusion antibody conjugate consisting of a fusion antibody and a cytotoxic drug, wherein the fusion antibody consists of two polypeptide chains:

[0046] (a) a first polypeptide chain comprising a VH, a CH1, a hinge, and a first Fc linked sequentially from the N-terminus to the C-terminus;

[0047] (b) a second polypeptide chain comprising a VL, a CL, a hinge, and a second Fc linked sequentially from the N-terminus to the C-terminus;

[0048] or,

[0049] (a) a first polypeptide chain comprising a VL, a CH1, a hinge, and a first Fc linked sequentially from the N-terminus to the C-terminus;

[0050] (b) a second polypeptide chain comprising a VH, a CL, a hinge, and a second Fc linked sequentially from the N-terminus to the C-terminus;

[0051] The Cys residue at position 214 of the second polypeptide chain CL is deleted (C214del, EU numbering, all numbers are in accordance with EU numbering below), or mutated to Ser (C214S), Gly (C214G), or Thr (C214T);

[0052] The cytotoxic drug is coupled to the Cys on the hinge of the first polypeptide chain and the second polypeptide chain.

[0053] In some embodiments, the cytotoxic drug is conjugated to Cys 226 and / or Cys 229 on the hinge of the first and second polypeptide chains.

[0054] The third aspect of the present invention provides a polynucleotide encoding the aforementioned fusion antibody.

[0055] The fourth aspect of the present invention provides a vector comprising a polynucleotide encoding a fusion antibody.

[0056] The fifth aspect of the present invention provides a host cell comprising a vector encoding the aforementioned fusion antibody polynucleotide.

[0057] The last aspect of the present invention provides a pharmaceutical composition comprising the aforementioned fusion antibody.

[0058] The present invention discovered that when the last Cys residue at the end of the second polypeptide chain CL is deleted (C214del) or mutated to serine (Ser) (C214S), and the Cys residue at position 220 is absent (C220del) or mutated to Ser (C220S) on the hinge of the first and second polypeptide chains, the formation of non-target bands (thioether products) that cannot be reduced by the fusion antibody under DTT reducing conditions can be effectively reduced. Furthermore, further deletion (C226del) or mutation of the second Cys residue to Ser (C226S) and / or deletion (C229del) or mutation of the third Cys residue to Ser (C229S) on the hinge of the first and second polypeptide chains can also effectively prevent the formation of non-target bands (thioether products).

[0059] The applicant further discovered that after opening the disulfide bonds on the hinge of the fusion antibody of the present invention using the reducing agent Tcep (tris(2-carbonylethyl)phosphine hydrochloride), a cytotoxic drug such as MMAE, Dxd, or DUPA was conjugated to the reduced thiol group of Cys on the hinge. The resulting conjugated product was structurally stable (see the bands between 130-250 kD for BEC8283 in FIG3F ). In contrast, conjugated products obtained using other commercially available ADC conjugation technologies exhibit multiple bands on a non-reducing SDS-ELISA (see lanes 2-3 for aHER2 mAb in FIG3F ). This suggests that the conjugates based on the fusion antibody of the present invention have greater structural stability. Furthermore, because the Cys involved in the conjugation are all located in the hinge region, the resulting conjugates exhibit more uniform DAR values, which helps ensure consistency in their clinical efficacy and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying drawings constituting this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0061] FIG1 is a schematic diagram of the structure of the fusion antibody of the present invention.

[0062] Figures 2A-2C are SDS-PAGE gel images of the fusion antibody of Example 2 after adding the reducing agent DTT. Lanes 1-10 in Figure 2A are MTE6263, MTE6465, MTE6667, MTE6869, MTE7071, MTE7273, MTE7475, MTE7677, MTE7879, and MTE8081, respectively; lanes 1-11 in Figure 2B are BEC2728, BEC8283, BEC2930, BEC3132, BEC3334, BEC3536, BEC3738, BEC3940, BEC4142, BEC4344, BEC4546; lanes 1-10 in Figure 2C are BEC9293, BEC9495, BEC9697, BEC9899, ​​BEC0001, BEC0203, BEC0405, BEC0607, BEC0809, and BEC1011, respectively; Figure 2D is an SDS-PAGE gel image after DTT reduction, lane 1 is PEC-3132, and lane 2 is PEC-9903; "-" in Figure 2E indicates no reducing agent DTT is added, and "+" indicates the addition of reducing agent DTT, where lane 1 is PEC-1415 and lane 2 is PEC-1516.

[0063] Figures 3A-3F are SDS-PAGE gel images of the fusion antibodies and their coupling products in Examples 2 and 3, "-" and "+" indicate no reducing agent and reducing agent DTT, respectively; Figure 3A shows the BEC9293 fusion antibody and its coupling product, wherein lane 1 is the BEC9293 fusion antibody, lane 2 is the BEC9293 fusion antibody after Tcep treatment, and lane 3 is the product after the BEC9293 fusion antibody is coupled to the molecule after Tcep treatment; Figure 3B shows the BEC9495 fusion antibody and its coupling product, wherein lane 1 is the BEC9495 fusion antibody, lane 2 is the BEC9495 fusion antibody after Tcep treatment, and lane 3 is the product after the BEC9495 fusion antibody is coupled to the DUPA molecule after Tcep treatment; Figure 3C shows the MTE6465 fusion antibody and its coupling product, wherein lane 1 is the MTE6465 fusion antibody, lane 2 is the MTE6465 fusion antibody after Tcep treatment, and lane 3 is the product after the BEC9495 fusion antibody is coupled to the DUPA molecule after Tcep treatment. Lane 3 shows the product of the MTE6465 fusion antibody after Tcep treatment coupled to the DUPA molecule; Figure 3D shows the MTE9091 fusion antibody and its coupling product, wherein lane 1 shows the MTE9091 fusion antibody, lane 2 shows the MTE9091 fusion antibody after Tcep treatment, and lane 3 shows the product of the MTE9091 fusion antibody after Tcep treatment coupled to DUPA; Figure 3E shows the MTE8687 fusion antibody and its coupling product, wherein 1 shows the MTE8687 fusion antibody, 2 shows the MTE8687 fusion antibody after Tcep treatment, and 3 shows the product of the MTE8687 fusion antibody after Tcep treatment coupled to the DUPA molecule; Figure 3F shows BEC8283 without the addition of the reducing agent DTT, aHER2 antibody and its coupling product, wherein 1 shows the sample treated with Tcep, 2 shows the product of the sample treated with Tcep coupled with Dxd, and 3 shows the product of the sample treated with Tcep coupled with MMAE.

[0064] 4A-4D are mass spectrometry assays of Dxd-based coupling products.

[0065] FIG5A and FIG5B show the binding activities of the fusion antibody of Example 3 to the antigens c-MET and EGFR, respectively, as detected by ELISA.

[0066] FIG6A-FIG6D show the inhibition of different cell signaling pathways by the fusion antibodies detected by WB.

[0067] FIG7A is a graph showing changes in tumor size in tumor-bearing mice after administration of bispecific antibodies and their conjugates, and FIG7B is a graph showing changes in body weight in tumor-bearing mice.

[0068] Detailed description of the invention

[0069] 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.

[0070] As used herein, "antibody fusion" refers to an antibody or antibody-based fusion protein composed of two different polypeptide chains, each comprising an Fc, wherein the Fc of one polypeptide chain forms an Fc dimer with the Fc of the other polypeptide chain, and the two polypeptide chains form at least one antigen binding domain.

[0071] As used herein, "Fc" is used to define the C-terminal domain of at least a portion of the constant region in an immunoglobulin heavy chain. It means a polypeptide comprising the constant region of an antibody (excluding the first constant region immunoglobulin domain). Therefore, Fc refers to the last two constant region immunoglobulin domains of human IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. Due to the homodimeric nature of molecules comprising the Fc region, the first Fc and the second Fc of the present invention comprise modified CH3 domains, wherein the modified CH3 domains comprise amino acid mutations that promote the formation of heterodimers of the first Fc and the second Fc (e.g., chimeric mutations, complementary mutations, locking and docking mutations, knobs into holes mutations, charge mutations, chain exchange engineered domain (SEED) mutations, etc.). Therefore, the first Fc and the second Fc can be selected from IgG, IgA, IgE, or IgM isotypes; further, the first Fc and the second Fc 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.

[0072] As used herein, "hinge" includes the upper hinge region and the lower hinge region of an antibody, which can be derived from IgA, IgB, IgM, IgE and IgG. When derived from IgG, it can be selected from IgG1, IgG2, IgG3 or IgG4. When selected from IgG1, its sequence is: EPKSCDKTHTCPPCP (E216-P230, EU numbering) (SEQ ID NO: 81), wherein: (1) C220 is deleted or mutated to another non-sulfhydryl amino acid; (2) C220 is deleted or mutated to another non-sulfhydryl amino acid, and C226 is retained, deleted or mutated to another non-sulfhydryl amino acid; (3) C220 is deleted or mutated to another non-sulfhydryl amino acid, C226 is retained, deleted or mutated to another non-sulfhydryl amino acid, and C229 is retained, deleted or mutated to another non-sulfhydryl amino acid; (4) C220 is deleted or mutated to another non-sulfhydryl amino acid, and C229 is retained, deleted or mutated to another non-sulfhydryl amino acid. When selected from IgG4, its sequence is: ESKYGPPCPPCP(E216-P230) (SEQ ID NO: 94). Example

[0073] The examples are for illustrative purposes only and are not intended to limit the present invention in any way.

[0074] Example 1 Vector Construction

[0075] The VL and VH gene segments that constitute the antibody and the gene segments of the antibody constant region were synthesized separately, and the above gene segments were amplified by PCR. The amplified VH gene segment was connected to the gene segment of the antibody constant region by overlap PCR as the first chain; the amplified VL gene segment was connected to the gene segment of the antibody constant region as the second chain, as shown in Figure 1; the above fragments were further connected to the pFuse vector (InvivoGen, CA) used for eukaryotic expression by 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:

[0076] Table 1 Fusion antibody sequences and hinges

[0077] Example 2 Expression and purification of fusion antibodies

[0078] 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 and purified using a GE AKTA chromatography system and a Superdex 200 Increase 10 / 300GL gel exclusion column in PBS buffer (pH 7.4). The purified sample was stored in PBS buffer (pH 7.4). The composition and purity of the fusion antibody were determined by SDS-PAGE under reducing and non-reducing conditions. The results are shown in Figures 2A-2D. In Figure 2A , lanes 2, 5, 8, and 9 under reducing conditions and lanes 2, 5, 6, and 8 in Figure 2B showed no extraneous bands, indicating high purity. However, the remaining lanes had obvious non-target bands at 130-250 kD, suggesting that mutating or deleting the first Cys on the hinge can effectively reduce the formation of non-target bands.

[0079] Example 3 Preparation of fusion antibody conjugated products

[0080] In this study, we selected three toxin molecules based on MMAE, Dxd, and Dupa for the preparation of conjugated products. A certain amount of the fusion antibody described in Example 2 was subjected to Cys-based site-directed conjugation. By optimizing the reduction reaction with tris(2-carbonylethyl)phosphine hydrochloride (TCEP, CAS: 51805-45-9), the interchain disulfide bonds of the antibody were opened. A payload with a maleimide linker (Payload 1, Deruxtecan, CAS: 1599440-13-7; Payload 2, vc-MMAE, CAS: 646502-53-6) was then added. An alkylation reaction occurred via nucleophilic Michael addition of the thiol group, forming a stable thioether bond and producing a relatively uniform conjugated product. The reduction reaction is to react the heterodimeric fusion protein and TCEP at a molar ratio of 1 / 20 in a 37°C incubator for 60 minutes. After the reaction, the sample is removed and placed on ice for cooling. Then, pre-cooled Payload with a linker is added at a molar ratio of 20 / 1 to the heterodimeric fusion protein. The reaction is carried out on ice for 2 hours. The TCEP introduced by the reduction reaction and the excess unreacted Payload are removed by a 40kDa Zeba desalting centrifugal column (CAS: 87766). At the same time, the coupled sample is replaced from the previous DPBS buffer system to a suitable buffer system (10 mg / mL sucrose, 20 mg / mL glycine, 1.47 mg / mL glutamate, pH 4.0). The coupled products were analyzed by SDS-PAGE for composition and purity under both reducing and non-reducing conditions. The results (see Figures 3A-3F) showed that the sizes of the fusion antibodies coupled to Dxd, MMAE, or DUPA were as expected in both non-reduced and reduced states, and no single chains of 55 kDa or 70 kDa were observed to fall out under non-reducing conditions, indicating that the structures of the fusion antibodies and their coupled products were intact under non-reducing conditions.

[0081] Example 4 Stability Test

[0082] The fusion antibody in Example 2 and the coupled product in Example 3 were mixed with freshly prepared thermal shift dye and shift buffer (Protein Thermal Shift TM Dye Kit, ThermoFisher Scientific, Cat. 4461146) were mixed according to the manufacturer's recommended ratio and the ViiA TMThermal scanning was performed at a heating rate of 0.05°C / s from 25°C to 99°C using a Real-Time PCR System or at a heating rate of 1.0°C / min from 20°C to 95°C using a Prometheus NT.48 Protein Stability Analyzer. Thermal melting temperatures (Tm) were 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.

[0083] The results are shown in Table 2. BEC8283 exhibits a high aggregation temperature, Tagg, similar to that of BEC2728 and higher than the Tagg values ​​of the other structures listed in the table, suggesting that deleting the first Cys residue in the hinge does not alter the protein's aggregation temperature, or affect its stability. Furthermore, BEC8283 exhibits a high initial melting temperature, Tm-1, similar to that of BEC2728.

[0084] The initial melting temperature (Tm-1) of MTE6465 is higher than that of all other structures listed (Table 3). BEC9495 and BEC0607 also have relatively high Tm-1 values, which are essentially consistent with those of BEC9293 (Table 4). This suggests that altering the first Cys position in the hinge has minimal effect on the thermal stability of the protein structure.

[0085] Table 2 Tm and Tagg values ​​of fusion antibodies

[0086] Table 3 Tm values ​​of fusion antibodies

[0087] Table 4 Tm values ​​of fusion antibodies

[0088] Table 5 Tm values ​​of fusion antibodies and conjugates

[0089] Example 5 Mass Spectrometry Analysis

[0090] The fusion antibody and its conjugated product from Example 3 were incubated with PNGase F (NEB) at a concentration of 1 mg / ml overnight at 37°C. The deglycosylated sample was reduced with 10 mM DTT and injected onto a 300SB-C8, 2.1 x 50 mm column in an HPLC-Q-TOF-MS (Agilent, USA). MS was performed, and the DAR value was calculated using the "Area Under Curve" (AUC) function in GraphPad Prism 7 software. As shown in Figure 4, the molecular weights of the fusion antibody and its conjugated product were generally consistent with theoretical predictions. Both chains of the Dxd-conjugated fusion antibody carried a specific number of small molecule drugs, which was equal to the number of reduced Cys residues in the hinge region, and the DAR value remained constant.

[0091] Example 6 Antigen Binding Activity Detection

[0092] 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 fusion 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 dilution gradients. Add 100 ul of the diluted fusion antibody and the corresponding control sample 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, 100 μl of freshly prepared TMB colorimetric reagent (BioLegend, Cat. 421101) was added to each well and incubated in the dark for 10 minutes at room temperature. Then, 100 μl of 1 M H₂SO₄ was added to each well and incubated for 2 minutes for color development. The results were read on a microplate reader at 450 nm. 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. As shown in Figure 5, the binding abilities of the fusion antibodies BEC8283 and BEC8485 to the target antigens cMet and EGFR were similar to those of the control.

[0093] Example 7 Phosphorylation Assay WB

[0094] 1 x 106 cells were seeded in a six-well plate and incubated at 37°C for 12 hours. The medium was then removed and replaced with serum-free medium for 24 hours. After starvation, the cells were treated with the fusion antibody sample, control sample, 50 ng / ml EGF, and 100 ng / ml HGF, respectively, and incubated at 37°C for 15 minutes. After incubation, the cells were washed with pre-chilled DPBS and then added with cell lysis buffer. After lysis on ice for 1 hour, the supernatant was collected by centrifugation. The supernatant was quantified using a BCA assay kit. Equal amounts of cell lysate were added with DTT-containing loading dye and heated at 95°C for 10 minutes. The samples were then loaded and run on an SDS-PAGE gel. The SDS-PAGE gel was transferred to a membrane using a transfer apparatus at low temperature. After transfer, the PVDF membrane was blocked at room temperature for 1 hour before incubation with the corresponding primary and secondary antibodies. After secondary antibody incubation, the cells were washed thoroughly, and substrate was added for color development and photographed.

[0095] The results are shown in Figures 6A-6D. In A549 cells, under EGF stimulation, the heterodimeric fusion protein completely inhibited EGFR phosphorylation and partially inhibited the phosphorylation of the proliferation signal ERK (Figure 6A). In the absence of EGF stimulation, the heterodimeric fusion protein still had a significant inhibitory effect on the phosphorylation of the proliferation signal ERK (Figure 6B). In the presence or absence of HGF induction, the heterodimeric fusion protein had a significant inhibitory effect on the phosphorylation of ERK and AKT. When HGF was added for induction, the heterodimeric fusion protein further inhibited the phosphorylation of cMET-1349 and cMET-1234 / 1235 (Figures 6C-6D).

[0096] Example 8 Pharmacokinetic Evaluation in Mice

[0097] 6-8 week old C57BL6J mice were administered via tail vein. The fusion antibody was administered only at a high dose of 10 mg / kg. The fusion antibody conjugate product was administered at three doses: 8 mg / kg, 4 mg / kg, and 1 mg / kg. Eye blood was collected at different time points. The concentrations of intact fusion antibody and its conjugate product in serum samples at each time point were determined by ELISA, and the data were processed using GraphPad Prism. As shown in the table, the fusion antibody conjugate product exhibited similar pharmacokinetic behavior to the fusion antibody.

[0098] Table 6 In vivo half-life of fusion antibodies and their conjugates

[0099] Example 9 Pharmacodynamic Evaluation in Mice

[0100] Nude mice aged 6-8 weeks were subcutaneously inoculated with tumor cells. 5x10e6 NCI-H1975 or BEC cells were inoculated on day 0. On day 8, when tumors were approximately 100-200 mm³, a single dose was administered via the tail vein. The fusion antibody conjugate product was administered at doses of 5, 2.5, and 1 mg / kg, and the corresponding fusion antibody was administered at doses of 5, 2.5, and 1 mg / kg. Tumor size was monitored three times weekly. As shown in Figure 7, the fusion antibody BEC8283 exhibited a significant tumor-suppressive effect in NCI-H1975 tumor-bearing mice in a dose-dependent manner. The fusion antibody BEC8283 conjugate product exhibited a superior tumor-suppressive effect compared to the fusion antibody.

Claims

1. A fusion antibody comprising: (a) a first polypeptide chain comprising a heavy chain variable region (VH), a heavy chain first constant region (CH1), a hinge region, and a first Fc region sequentially connected from N-terminus to C-terminus; (b) a second polypeptide chain, comprising a light chain variable region (VL), a light chain constant region (CL), a hinge region, and a second Fc region sequentially connected from N-terminus to C-terminus; in, The cysteine (Cys) at position 214 of the second polypeptide chain CL is deleted (C214del, EU numbering, hereinafter all EU numbering is used); The VH and the VL constitute a first antigen-binding domain.

2. The fusion antibody of claim 1, wherein the hinges of the first polypeptide chain and the second polypeptide chain are different.

3. The fusion antibody according to claim 2, wherein the hinge between the first polypeptide chain and the second polypeptide chain does not contain Cys at position 220, i.e., C220del.

4. The fusion antibody of claim 3, wherein the first polypeptide chain has a hinge as shown in EPKSDKTHTCPPCP (SEQ ID NO: 82), and the second polypeptide chain has a hinge as shown in DKTHTCPPCP (SEQ ID NO: 101). The fusion antibody according to claim 1 , wherein the hinge of the first polypeptide chain and the second polypeptide chain are the same.

6. The fusion antibody of claim 5, wherein the hinge of the first polypeptide chain and the second polypeptide is selected from IgG1, the hinge is EPKSCDKTHTCPPCP(E216-P230) (SEQ ID NO: 81), and one or more Cys on the hinge is deleted and / or mutated to serine (Ser), threonine (Thr) or glycine (Gly).

7. The fusion antibody according to claim 6, wherein The first Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C220del) or mutated to Ser (C220S).

8. The fusion antibody of claim 7, wherein the second Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C226del) or mutated to Ser (C226S).

9. The fusion antibody of claim 7, wherein the third Cys on the hinge of the first polypeptide chain and the second polypeptide chain is deleted (C229del) or mutated to Ser (C229S).

10. The fusion antibody according to any one of claims 1 to 9, wherein The first Fc and the second Fc are derived from IgG1, IgG2, IgG3 or IgG4; The first Fc and the second Fc comprise amino acid substitutions that promote heterologous pairing between the first Fc and the second Fc. The fusion antibody according to claim 10 , wherein: One of the first Fc and the second Fc comprises an amino acid substitution of T366W, and the other of the first Fc and the second Fc comprises amino acid substitutions of T366S, L368A, and Y407V; One of the first Fc and the second Fc comprises amino acid substitutions T366W, S354C, and the other of the first Fc and the second Fc comprises amino acid substitutions T366S, L368A, Y407V, and Y349C; or 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.

12. The fusion antibody of any one of claims 1 to 10, wherein the N-terminus of one of the first polypeptide chain and the second polypeptide chain is further fused to an antibody heavy chain variable region (VH2), and the N-terminus of the other of the first polypeptide chain and the second polypeptide chain is further fused to an antibody light chain variable region (VL2), and VH2 and VL2 form a second antigen-binding domain.

13. The fusion antibody of any one of claims 1 to 12, wherein the fusion antibody comprises a first polypeptide chain and a second polypeptide chain consisting of the following amino acid sequences: SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 63 and SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66; SEQ ID NO: 67 and SEQ ID NO: 68; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 71 and SEQ ID NO: 72; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 79 and SEQ ID NO:

80. NO:80; SEQ ID NO:106 and SEQ ID NO:107; SEQ ID NO:107 and SEQ ID NO:108; SEQ ID NO:109 and SEQ ID NO:110; SEQ ID NO:111 and SEQ ID NO:

112.

14. A conjugate comprising the fusion antibody according to any one of claims 1 to 13, and a cytotoxic drug conjugated to Cys on the hinge of the fusion antibody.

15. A polynucleotide encoding the fusion antibody according to any one of claims 1 to 13.

16. A vector comprising the polynucleotide according to claim 15.

17. A host cell comprising a vector encoding the gene of claim 16.

18. A pharmaceutical composition comprising the fusion antibody according to any one of claims 1 to 13 or the conjugate according to claim 14.

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