Antibody-drug conjugate and preparation method therefor and use thereof

Abstract

The present disclosure relates to an anti-VEGF antibody-drug conjugate and a use thereof, and in particular to an anti-VEGF antibody-drug conjugate exhibiting antitumor activity. The present disclosure further comprises a composition comprising the antibody-drug conjugate and a method of using the composition.

Description

Antibody drug conjugates and preparation methods and uses thereof Technical Field

[0001] The present disclosure belongs to the field of medical technology and relates to various antibodies, antibody-drug conjugates and methods for preparing the same, as well as their use in preventing and / or treating diseases associated with abnormal cell activity, including but not limited to preventing and / or treating tumor diseases. Background Art

[0002] Under normal physiological conditions, angiogenesis is in a dynamic regulatory equilibrium, strictly controlled by a complex interaction of a series of factors that play a crucial role in angiogenesis during embryonic development. However, tumors can activate the "angiogenic switch," disrupting the dynamic balance of angiogenesis and forming tumor vessels with abnormal structure and function, accelerating tumor initiation, growth, and metastasis. Anti-angiogenesis and vascular normalization therapies have become landmark new strategies in cancer treatment.

[0003] Bevacizumab (trade name: Avastin) is a humanized monoclonal IgG1 antibody that binds to VEGF-A and blocks the VEGF / VEGFR signaling pathway, thereby inhibiting angiogenesis and inducing tumor vascular regression. In addition, low doses of bevacizumab can cause a transient normalization phase of tumor blood vessels. Currently, bevacizumab has been approved for use in combination with chemotherapy for the treatment of a wide range of solid tumor types, including colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). However, the off-target effects of chemotherapy drugs can lead to increased systemic toxicity and drug resistance, increase the patient's suffering during treatment, and severely limit the clinical application of monoclonal antibody / chemotherapeutic drug combinations. Therefore, there is an urgent need to find a treatment option that can break through the limitations of drug combinations.

[0004] ADCs are conjugates of antibodies and small molecule drugs, combining the targeting properties of antibodies with the activity of bioactive molecules, creating a biological missile with highly promising efficacy and safety advantages. Antibodies guide the ADC to bind to target cells, where it is then internalized. The small molecule drug is then released through enzymatic degradation by specific enzymes, treating the disease. ADCs have developed rapidly in recent years, with 17 ADCs already on the market. However, these currently marketed ADCs bind to tumor cell surface antigens through the antibody within the ADC. The toxin is then cleaved from the ADC after endocytosis, generating anti-tumor activity. However, ADCs targeting free antigens differ from traditional endocytic ADCs, making their development challenging. For the free antigen VEGF, developing an ADC specifically for free VEGF would provide cancer patients with a wider range of treatment options and offer broad market prospects.

[0005] Although there are reports in the prior art of VEGF ADC (Bevacizumab Vedotin) using MMAE as a toxin (Yanchen Li, et al., Bioorg Chem. 2023 Aug: 137: 106575. doi: 10.1016 / j.bioorg.2023.106575.), ADC using MMAE as a toxin exhibits unique toxicities such as ocular and peripheral neuropathy in clinical practice.

[0006] Therefore, there is an urgent need in the art to develop an antibody-drug conjugate targeting VEGF. Summary of the Invention

[0007] The present disclosure provides an anti-VEGF antibody-drug conjugate and its use in treating cancer.

[0008] The present disclosure provides a VEGF-ADC with a different mechanism of action, which has good anti-tumor activity and / or safety, broadens the range of clinical drug options, and has significant clinical value.

[0009] This disclosure discloses the use of camptothecin molecules as toxins for VEGF-ADC for the first time. It is an ADC with a new mechanism of action, has good anti-tumor activity, and has good systemic circulation stability and / or safety, thereby improving the therapeutic window.

[0010] The antibody-drug conjugate disclosed herein is the world's first ADC molecule targeting VEGF that is about to enter clinical trials. It has the effect of exerting the anti-angiogenesis effect of anti-VEGF antibodies, and can also simultaneously utilize the toxins (camptothecins) carried by the ADC to kill tumor cells. The two also have a synergistic anti-tumor effect. At the same time, it also utilizes the specific release mechanism of the ADC molecule disclosed herein in the tumor microenvironment. These multiple mechanisms of action can better inhibit tumors, thereby exerting two anti-tumor mechanisms through a single administration, which is also extremely convenient for patients.

[0011] Antibody Drug Conjugates

[0012] In a first aspect, the present disclosure provides an antibody drug conjugate, which is a compound of formula (I), or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug, or pharmaceutically acceptable salt or solvate (such as a hydrate) thereof:

[0013] Ab-[LD]q

[0014] Formula (I)

[0015] in,

[0016] Ab is an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF);

[0017] L is a bond or linking molecule that covalently links Ab and D;

[0018] q is an integer between 1 and 16 and represents the number of LDs covalently linked to the Ab;

[0019] D is a camptothecin drug molecule.

[0020] Ab

[0021] In some embodiments, Ab is an antibody or antigen-binding fragment thereof that specifically binds to VEGF (VEGF antibody or antigen-binding fragment thereof). In some embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three CDR-Hs, namely CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises three CDR-Ls, namely CDR-L1, CDR-L2, and CDR-L3.

[0022] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact schemes, or any combination thereof.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Chothia scheme.

[0024] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the definition of the Chothia scheme,

[0025] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:5;

[0026] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 10;

[0027] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17;

[0028] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23;

[0029] CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and

[0030] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0031] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the definition of the Chothia scheme,

[0032] CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:5;

[0033] CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 10;

[0034] CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17;

[0035] CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23;

[0036] CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and

[0037] CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the AbM scheme.

[0039] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as an Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the AbM protocol definition,

[0040] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:6;

[0041] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0042] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17;

[0043] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23;

[0044] CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and

[0045] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0046] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as an Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the AbM protocol definition,

[0047] CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:6;

[0048] CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 11;

[0049] CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17;

[0050] CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23;

[0051] CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and

[0052] CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Kabat scheme.

[0054] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the Kabat scheme definition,

[0055] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:7;

[0056] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 12;

[0057] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17;

[0058] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23;

[0059] CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and

[0060] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0061] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the Kabat scheme definition,

[0062] CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:7;

[0063] CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 12;

[0064] CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17;

[0065] CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23;

[0066] CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and

[0067] CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22.

[0068] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Contact scheme.

[0069] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the definition of the Contact scheme,

[0070] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:8;

[0071] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0072] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 15;

[0073] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:24;

[0074] CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 25; and

[0075] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:21.

[0076] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the definition of the Contact scheme,

[0077] CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:8;

[0078] CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 13;

[0079] CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 15;

[0080] CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:24;

[0081] CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 25; and

[0082] CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:21.

[0083] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the IMGT scheme.

[0084] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as an Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the IMGT protocol definition,

[0085] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:9;

[0086] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 14;

[0087] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 16;

[0088] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 20;

[0089] CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 18; and

[0090] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0091] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; or the VEGF antibody or antigen-binding fragment thereof as an Ab comprises three CDR-H CDR-H1, CDR-H2 and CDR-H3 and three CDR-L CDR-L1, CDR-L2 and CDR-L3, wherein, according to the IMGT protocol definition,

[0092] CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:9;

[0093] CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 14;

[0094] CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 16;

[0095] CDR-L1 consists of the amino acid sequence shown in SEQ ID NO: 20;

[0096] CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 18; and

[0097] CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as an Ab comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 in the following Chothia, AbM, Kabat, IMGT, or Contact definition schemes:

[0099] and

[0100] In some embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:3, and / or the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4.

[0101] In some embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein

[0102] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; or

[0103] (ii) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 4.

[0104] In some embodiments, Ab comprises a heavy chain variable region and a light chain variable region, wherein

[0105] (i) the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4; or

[0106] (ii) the heavy chain variable region comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.

[0107] In some embodiments, Ab comprises: a heavy chain variable region (VH) of SEQ ID NO: 3 and a light chain variable region (VL) of SEQ ID NO: 4.

[0108] In some embodiments, the Ab comprises a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:3, and a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:4.

[0109] In some embodiments, the Ab comprises a heavy chain variable region (VH) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:3, and a light chain variable region (VL) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:4.

[0110] In some embodiments, Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, the heavy chain variable region and the heavy chain constant region; and the light chain comprises, or consists of, the light chain variable region and the light chain constant region.

[0111] In some embodiments, the heavy chain constant region is a heavy chain constant region of (human) IgG, such as a heavy chain constant region of (human) IgG1, IgG2, IgG3 or IgG4. In some embodiments, the light chain constant region is a light chain constant region of (human) Kappa or Lambda.

[0112] In some embodiments, Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, and / or the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0113] In some embodiments, Ab comprises a heavy chain and a light chain, wherein

[0114] (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2; or

[0115] (ii) the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain consists of the amino acid sequence shown in SEQ ID NO: 2.

[0116] In some embodiments, Ab comprises a heavy chain and a light chain, wherein

[0117] (i) the heavy chain comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that has 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2; or

[0118] (ii) the heavy chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.

[0119] In some embodiments, the Ab comprises: a heavy chain of SEQ ID NO: 1 and a light chain of SEQ ID NO: 2.

[0120] In some embodiments, the Ab comprises a heavy chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 1, and a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:2.

[0121] In some embodiments, the Ab comprises a heavy chain that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO: 1, and a light chain that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:2.

[0122] In some embodiments, the Ab comprises two heavy chains and two light chains, eg, two identical heavy chains and two identical light chains.

[0123] In some embodiments, Ab is a humanized antibody, a human antibody, or a chimeric antibody.

[0124] In some embodiments, Ab is a full-length antibody.

[0125] In some embodiments, Ab is an antigen-binding fragment, such as single-chain Fv (scFv), Fab, Fab', F(ab')2, disulfide-linked Fv (sdFv), Fv, di-scFv, etc.

[0126] In some embodiments, Ab is selected from the antibodies or antigen-binding fragments thereof disclosed in WO9845331 or CN109053895B, such as Bevacizumab or a biosimilar thereof or an antigen-binding fragment thereof disclosed therein.

[0127] In some embodiments, the Ab is selected from Bevacizumab or a biosimilar thereof or an antigen-binding fragment thereof.

[0128] In some embodiments, Ab is selected from any one of the following Bevacizumab biosimilars or antigen-binding fragments thereof, including but not limited to ABP 215, BCD-021, FKB238, Mvasi (Bevacizumab-awwb), PF-06439535, SB8, Zirabev (Bevacizumab-bvzr), QL1101, IBI305, SHR-1603, BAT1706, HS008, MIL60 or an antigen-binding fragment thereof.

[0129] L

[0130] In some embodiments, L comprises an amino acid residue or a short peptide consisting of 2-10 amino acid residues.

[0131] In some embodiments, L comprises an amino acid residue that can be cleaved in the tumor microenvironment or a short peptide consisting of 2-10 amino acid residues.

[0132] In some embodiments, the amino acid residues in L are natural amino acid residues or modifications thereof.

[0133] In some embodiments, L comprises a moiety selected from the group consisting of Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA 1or 2-10 amino acid residues selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AA 1 A short peptide composed of amino acid residues, where AA 1 Selected from Understandable, AA 1 of and Indicates that the bond is connected to the rest of the molecule.

[0134] In some embodiments, L comprises Val-AA 1 -Gly, where AA 1 As defined herein.

[0135] In some embodiments, L is selected from Position 1 is connected to Ab and position 2 is connected to D.

[0136] D

[0137] In some embodiments, D is selected from camptothecin, SN38, DXD, a substituent-modified camptothecin, a substituent-modified DXD, or a substituent-modified SN38.

[0138] In some embodiments, D is selected from:

[0139] One of them is connected to L.

[0140] q

[0141] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0142] LD

[0143] In some embodiments, LD is selected from the group consisting of:

[0144] One of them is connected to Ab.

[0145] ADC

[0146] In some embodiments, the antibody drug conjugate or compound of formula (I) is selected from

[0147] wherein q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0148] It can be understood that S in the structural formula is derived from Ab, that is, the -LD moiety is covalently linked to Ab via the sulfur (S) atom derived from Ab.

[0149] In some embodiments, the Ab is a VEGF antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof as defined in the "Ab" section herein.

[0150] In some embodiments, the VEGF antibody or antigen-binding fragment thereof as an Ab comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact schemes, or any combination thereof.

[0151] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to VEGF as Ab comprises: a heavy chain variable region (VH) of SEQ ID NO: 3 and a light chain variable region (VL) of SEQ ID NO: 4. Preferably, Ab is selected from Bevacizumab or a biosimilar thereof or an antigen-binding fragment thereof.

[0152] In some embodiments, q is selected from 2, 4, 6, or 8.

[0153] In some embodiments, q is selected from 4 or 8.

[0154] In some embodiments, the antibody drug conjugate or compound of formula (I) is selected from:

[0155] Wherein Ab is selected from Bevacizumab or its biosimilars.

[0156] Antibody Drug Conjugate Group

[0157] In the second aspect of the present disclosure, the present disclosure provides a population of antibody-drug conjugates, comprising or consisting of the antibody-drug conjugate according to the first aspect or its tautomers, stereoisomers, isotopic derivatives (such as deuterated derivatives), prodrugs or pharmaceutically acceptable salts or solvates, wherein the antibody-drug conjugates have one, two or more q values.

[0158] In some embodiments, the q value and the average DAR are close when one q value of the antibody drug conjugates in the population of antibody drug conjugates accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99% or more).

[0159] In some embodiments, when there is only one q-valued ADC in the population of ADCs, the q-value and the average DAR are equal.

[0160] In some embodiments, when the antibody drug conjugates of the antibody drug conjugate population have two or more q values, the proportion of antibody drug conjugates with a particular q value in all antibody drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%.

[0161] In some embodiments, the average drug to antibody ratio (average DAR) in the antibody drug conjugate population is an integer or decimal selected from 1-16, preferably 1-10.

[0162] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5.

[0163] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from about 2.0, about 4.0, about 6.0, or about 8.0.

[0164] In some embodiments, the average drug to antibody ratio (average DAR) in the antibody drug conjugates in the population of antibody drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0165] In some embodiments, the antibody drug conjugate population contains ADCs having a distribution of DARs of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., a drug loading population of 1.5, 2, 4, 6, and 8). It is worth noting that degradation products can be produced such that the mixture may also contain DARs of 1, 3, 5, and 7. In addition, the antibody drug conjugate population may also have an average DAR greater than 8. The antibody drug conjugate is produced by reducing interchain disulfides and then conjugating. In some embodiments, the antibody drug conjugate comprises both: an antibody drug conjugate having a DAR of 4 or less (i.e., a drug loading population of 4 or less) and an antibody drug conjugate having a DAR of 6 or more (i.e., a drug loading population of 6 or more).

[0166] Pharmaceutical composition

[0167] In the third aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate, or a group of antibody-drug conjugates of the second aspect, and optionally one or more pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0168] In certain embodiments, the pharmaceutical composition comprises an effective amount of the antibody drug conjugate of the first aspect or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate thereof, or a population of the antibody drug conjugates of the second aspect.

[0169] In certain embodiments, the pharmaceutical composition comprises the above-mentioned antibody-drug conjugate or its tautomers, stereoisomers, isotopic derivatives (such as deuterated derivatives), prodrugs or pharmaceutically acceptable salts or solvates, and pharmaceutical excipients.

[0170] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise the population of antibody drug conjugates of the second aspect, and pharmaceutically acceptable excipients.

[0171] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise the antibody drug conjugate of the present disclosure or its tautomers, stereoisomers, isotopic derivatives (such as deuterated derivatives), prodrugs or pharmaceutically acceptable salts or solvates, and pharmaceutical excipients.

[0172] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or antibody-drug conjugate population is an integer or decimal selected from 1-10.

[0173] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5.

[0174] In some embodiments, the DAR in the pharmaceutical composition or antibody-drug conjugate population is selected from the group consisting of: 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, 8±0.5.

[0175] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from about 2.0, about 4.0, about 6.0, or about 8.0.

[0176] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0177] In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate of the first aspect or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate and a buffer.

[0178] In some embodiments, the pharmaceutical composition comprises a population of antibody drug conjugates of the second aspect and a buffer.

[0179] In some embodiments, the pharmaceutical composition is used for therapy. In some embodiments, the pharmaceutical composition is used to treat and / or prevent diseases associated with abnormal cell activity (e.g., tumors). In some embodiments, the pharmaceutical composition is used to modulate (inhibit or block) the activity of VEGF. In some embodiments, the pharmaceutical composition is used to treat or prevent diseases associated with the activity of VEGF or diseases associated with the targets of VEGF. In some embodiments, the pharmaceutical composition is used to treat or prevent tumors associated with the activity of VEGF.

[0180] Uses in tumor treatment

[0181] In a fourth aspect, the present disclosure provides uses of the disclosed substances (including the aforementioned antibody drug conjugates, groups of antibody drug conjugates, and the aforementioned pharmaceutical compositions) for use as drugs or for preparing drugs, wherein the drugs are used for therapy, for example, for treating and / or preventing diseases associated with abnormal cell activity (e.g., cancer diseases). In some embodiments, the antibody drug conjugates, groups of antibody drug conjugates, and the aforementioned pharmaceutical compositions are in an effective amount, for example, a prophylactic or therapeutically effective amount.

[0182] In some embodiments, the antibody-drug conjugate or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate of the first aspect of the present disclosure, the population of antibody-drug conjugates of the second aspect, and the use of the pharmaceutical composition of the third aspect are provided for use as a drug or for the preparation of a drug for modulating (inhibiting or blocking) the activity of VEGF.

[0183] In some embodiments, the antibody-drug conjugate or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate of the first aspect of the present disclosure, the population of antibody-drug conjugates of the second aspect, and the use of the pharmaceutical composition of the third aspect are provided for use as a drug or for preparing a drug for treating or preventing a disease related to the activity of VEGF or a disease related to the target of VEGF.

[0184] In some embodiments, the antibody-drug conjugate or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate of the first aspect of the present disclosure, the population of antibody-drug conjugates of the second aspect, and the use of the pharmaceutical composition of the third aspect are provided for use as a drug or for the preparation of a drug for treating or preventing tumors associated with the activity of VEGF.

[0185] Methods for treating tumors

[0186] In a fifth aspect, the present disclosure provides a method for treating and / or preventing diseases associated with abnormal cell activity (e.g., tumors) by using the antibody-drug conjugate of the first aspect or its tautomer, stereoisomer, isotopic derivative (e.g., deuterated derivative), prodrug, or pharmaceutically acceptable salt or solvate, the group of antibody-drug conjugates of the second aspect, or the pharmaceutical composition of the third aspect.

[0187] In some embodiments, provided are methods for using the antibody-drug conjugate of the first aspect, or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug, or pharmaceutically acceptable salt or solvate, the population of antibody-drug conjugates of the second aspect, or the pharmaceutical composition of the third aspect, to modulate (inhibit or block) the activity of VEGF.

[0188] In some embodiments, methods are provided for treating or preventing a disease associated with VEGF activity or a disease associated with a VEGF target by using the antibody-drug conjugate of the first aspect, or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug, or pharmaceutically acceptable salt or solvate thereof, the population of antibody-drug conjugates of the second aspect, or the pharmaceutical composition of the third aspect. In some embodiments, the disease is a tumor associated with VEGF activity.

[0189] In some embodiments, the method comprises administering the antibody drug conjugate of the first aspect or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate, the population of antibody drug conjugates of the second aspect, or the pharmaceutical composition of the third aspect.

[0190] In the third, fourth, and fifth aspects, the diseases associated with VEGF activity, VEGF target sites, or abnormal cell activity include tumors, such as cancer. The cancer may be in the early, middle, or late stages, or may be metastatic. In some embodiments, the cancer may be a solid tumor or a hematologic tumor.

[0191] In one embodiment, the tumor refers to elevated protein levels (e.g., expression) of VEGF, or elevated nucleic acid levels of VEGF in tumor tissue or tumor cells of the individual, e.g., compared to adjacent normal tissue or normal cells (e.g., normal cells in a tissue) of the individual, or the same tissue or cells therein of a healthy individual.

[0192] The tumor is selected from, but not limited to, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma), colon cancer (such as human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (such as colorectal adenocarcinoma).

[0193] It will be appreciated that the various aspects of the present disclosure and their various embodiments and features can be combined with any other aspects, embodiments and features to provide other embodiments, and the embodiments of these combinations also fall within the scope of the present application. In addition, the methods, examples and materials described herein are merely illustrative and do not limit the present disclosure in any way. Changes that are readily apparent to those skilled in the art will be included within the purpose and scope of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0194] definition

[0195] The terms and symbols used in this application have the meanings commonly understood by those skilled in the art, unless otherwise specified. In particular, the following terms in this application have the meanings indicated below, unless otherwise specified.

[0196] As used herein, the expressions "a," "an," "the," "said" or the entity itself include both the singular and the plural forms unless otherwise indicated.

[0197] The immunoglobulin molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin. Preferably, the antibodies disclosed herein comprise or consist of a VH region, a VH CDR (herein often referred to as CDR-H), a VL region, or a VL CDR (herein often referred to as CDR-L) having any of the amino acid sequences described in the Sequence and Detailed Information Tables, or a fragment or variant thereof.

[0198] Preferably, the antibodies disclosed herein comprise or consist of a VH domain, a VH CDR (herein often represented by CDR-H), a VL domain, or a VL CDR (herein often represented by CDR-L) having any of the amino acid sequences described in the Sequence and Specific Information Tables, or a fragment or variant thereof.

[0199] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. The modifier "monoclonal" herein indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies and is not to be construed as requiring production by a particular method.

[0200] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity. Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0201] The term "antigen-binding fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region fragments, diabodies, linear antibodies and single-chain antibody molecules. The term "antigen-binding fragment" as used herein refers to a partial fragment of an antibody with antigen-binding activity, wherein the fragment has all or part of the function of an antibody, including but not limited to single-chain Fv (scFv), Fab, Fab', F(ab')2, disulfide-linked Fv (sdFv), Fv, di-scFv, etc. The term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, the term is not limited to these molecules, as long as the fragment has binding affinity to the antigen. In addition, these functional fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0202] The term "Fab'" as used herein refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, the Fab' disclosed herein also includes Fab' produced using genetically modified antibody genes.

[0203] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are linked by a linker or directly (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0204] The terms "variable region," "variable domain," or "variable domain" refer to the domains of an antibody's heavy and / or light chains involved in antigen binding. Natural IgG antibodies, VH and VL, each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The terms "complementarity-determining regions" or "CDRs" refer to the regions within the variable domain that primarily contribute to antigen binding; "framework" or "FRs" refers to the variable domain residues excluding the CDR residues. The VH contains three CDR regions: CDR-H1, CDR-H2, and CDR-H3; the VL contains three CDR regions: CDR-L1, CDR-L2, and CDR-L3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0205] The boundaries of the amino acid sequence of the CDR can be determined by various well-known definition schemes, for example: the "Kabat" definition scheme rules (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" definition scheme, the "ABM" definition scheme, the "contact" definition scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and the ImMunoGenTics (IMGT) definition scheme (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between the various definition schemes is well known to those skilled in the art and is exemplified as shown below.

[0206] CDR defines the relationship between the schemes

[0207] It should be understood that the antibodies or antigen-binding fragments thereof described herein may be modified to a certain extent, such as by amino acid substitution, deletion and / or addition (preferably outside the CDR region, more preferably outside the variable region) to obtain antibodies that still have the activity of binding to the antigen, and these antibodies are also encompassed within the anti-VEGF antibodies described herein.

[0208] The term "complete antibody" or "full-length antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. A complete antibody is preferably a complete antibody with one or more effector functions. In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of a human immunoglobulin are also replaced with non-human residues. Moreover, humanized antibodies may also comprise residues that are not present in the recipient antibody or the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally comprise at least one, usually two variable regions, in which all or nearly all of the hypervariable loops correspond to those of non-human immunoglobulins, while the FRs are entirely or almost entirely human immunoglobulin sequences. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc, usually a human immunoglobulin Fc).

[0209] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0210] In this article, the CDRs contained in the antibodies or antigen-binding fragments thereof disclosed herein can be determined according to various definition schemes known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof disclosed herein are preferably determined by the Kabat, Chothia or AbM, IMGT, Contact definition schemes, respectively.

[0211] As used herein, the term "framework residue region" or "FR residues" refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0212] The compilation of the twenty conventional amino acids involved herein follows conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; and glutamine can be represented by Q or Gln.

[0213] The "Vascular Endothelial Growth Factor" (VEGF), described herein, is a signaling protein produced by various cells that stimulates blood vessel formation. Specifically, VEGF is a subfamily of platelet-derived growth factors, belonging to the cysteine ​​knot growth factor family. They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the formation of new blood vessels from existing vasculature). In mammals, the VEGF family includes five members: VEGF-A, placental growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D. Of these members, VEGF-A was the first to be discovered. Therefore, VEGF-A may also be referred to as VEGF. Its activity has primarily been studied on vascular endothelial cells, although it also affects many other cell types (for example, stimulating the migration of monocytes / macrophages, neurons, cancer cells, and renal epithelial cells). In vitro studies have demonstrated that VEGF-A can promote the proliferation and migration of endothelial cells. In addition, VEGF-A also has the effect of vasodilation and increases the permeability of microvessels, which is why it was originally called vascular permeability factor. Herein, VEGF can be VEGF-A, such as human VEGF-A. In some embodiments, the VEGF-A is a VEG165 isoform, such as human VEGF-165.

[0214] As used herein, the term "antibody that specifically binds to VEGF" or "anti-VEGF antibody," "anti-VEGF," "VEGF antibody," or "antibody against VEGF" refers to an antibody that, or an antigen-binding fragment thereof, binds to VEGF protein with sufficient affinity.

[0215] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. In some embodiments, the amino acid is a natural amino acid, i.e., an amino acid that is naturally synthesized in organisms and participates in protein construction. The names of exemplary natural amino acids and their corresponding abbreviations are listed in the table below:

[0216] The amino acids disclosed herein also include other amino acids such as citrulline (Cit; C), which is also a naturally occurring amino acid.

[0217] Unless otherwise specified, the amino acids disclosed herein refer to L-amino acids.

[0218] The amino acids described herein may also be modified amino acids, where the modification can be accomplished by chemical or biological methods to modify the structure of a natural amino acid, thereby imparting new functions or properties. Such modifications can occur at sites such as the side chain, amino group, or carboxyl group of the amino acid.

[0219] As used herein, the term "a natural amino acid residue or a modification thereof" encompasses natural amino acids as well as modified amino acids.

[0220] A biosimilar refers to a biological product that is identical in sequence to an approved original biological drug (reference product, such as Bevacizumab or Avastin) and is highly similar in structure, function, quality, safety and efficacy, but minor differences (due to different production processes) are allowed.

[0221] As used herein, the term "immunoconjugate" refers herein to the attachment of an effector molecule to an antibody or its antigen-binding fragment via a linker, such that the antibody or its antigen-binding fragment can serve as a carrier for targeted transport of the effector molecule to a target site. The term "effector molecule" refers to the active portion of an antibody or antibody fragment conjugated to the present disclosure, and may include any portion for attaching an antibody or antibody fragment. In some embodiments, the effector molecule may be a drug such as a small molecule drug, DNA, RNA, enzyme, or polypeptide, etc. In some embodiments, the immunoconjugate encompasses an antibody-drug conjugate (ADC). Suitable effector molecules or active portions for attachment to antibodies include, for example, antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof. In some embodiments, the immunoconjugate of the present disclosure is an antibody-drug conjugate, i.e., ADC.

[0222] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present disclosure include immune checkpoint inhibitors or immune checkpoint agonists.

[0223] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., tumor and infectious disease) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0224] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human. In certain embodiments, the subject (e.g., a human) suffers from a tumor and an infectious disease, or has the risk of suffering from the above diseases.

[0225] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., tumors and infectious diseases) refers to an amount sufficient to prevent, stop or delay the occurrence of a disease (e.g., tumors and infectious diseases); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0226] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0227] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0228] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0229] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0230] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the antibodies disclosed herein also encompass variants of the antibody molecules that have a considerable degree of identity, for example, at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes.

[0231] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0232] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0233] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0234] When referring to "agents of the disclosure" or "agents of the invention", this encompasses one or more of the various molecules or compositions defined herein, for example, encompasses one or more of the following: an antibody drug conjugate as defined herein, or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), prodrug or pharmaceutically acceptable salt or solvate thereof, a population of antibody drug conjugates, or a pharmaceutical composition.

[0235] The term "pharmaceutically acceptable" means that when the molecule itself, molecule fragment or composition is appropriately administered to an animal or human, it does not produce adverse, allergic or other untoward reactions. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0236] The term "drug to antibody ratio" or "DAR" refers to the ratio of the number of drug moieties (D) coupled to an antibody portion (Ab) as described herein to the number of antibody portions. The DAR of an ADC can range from 1 to 20, but higher loadings are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of drug moieties (D) coupled to the Ab portion as described herein to the Ab portion in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the antibody drug conjugates of the present disclosure is 1.0-20.0, e.g., 1.0-18.0, 1.0-16.0, 2.0-14.0, 3.0-12.0, 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, e.g., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 12.0, and 16.0, and ranges having two of these values ​​as endpoints.

[0237] In the process of determining the DAR value by mass spectrometry, the antibody has been reduced to separated heavy and light chains. DAR1 represents a conjugate containing a light chain or heavy chain coupled to one toxin molecule; DAR2 represents a conjugate containing a light chain or heavy chain coupled to two toxin molecules; DAR3 represents a conjugate containing a light chain or heavy chain coupled to three toxin molecules; and so on.

[0238] The term "drug" refers to chemical substances that can alter or identify physiological functions and pathological states of the body and can be used to prevent, diagnose, and treat disease. This includes substances that inhibit or prevent cellular function and / or cause cell death or destruction. Drugs include cytotoxic agents, particularly small molecule cytotoxic agents. There is no strict distinction between drugs and poisons. Poisons are chemical substances that can have toxic effects on the body and harm human health even at relatively low doses. Excessive doses of any drug can produce toxic reactions.

[0239] Cytotoxic agents are substances that inhibit or prevent cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic agents include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 13 1.I 125 、Y 90 、Re 186 、Re 188 、Sm 15 3. Bi 212 、P 32 and radioisotopes of Lu), chemotherapeutic drugs, antibiotics and nucleolytic enzymes. In addition, it includes but is not limited to DNA topoisomerase inhibitors (e.g., camptothecin-type bioactive molecules, such as camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or microtubule inhibitors (e.g., MMAF-type microtubule inhibitors, MMAE-type microtubule inhibitors).

[0240] For the avoidance of doubt, the term "drug" that can be a component of an ADC does not refer solely to "drugs" approved by pharmaceutical regulatory authorities. It also includes any compound with potential therapeutic biological activity in clinical practice, R&D, or academic research. Furthermore, it should be understood that this term differs from the meaning of "drug for use in the preparation of a medicine." It should be understood that drug molecules may need to be functionalized or derivatized in order to be attached to a linker, and the resulting compounds are also included within the scope of the drugs disclosed herein.

[0241] As used herein, "substituent-modified camptothecin," "substituent-modified DXD," and "substituent-modified SN38" refer to those chemically modified at any available position on the camptothecin, DXD, or SN38 molecule, respectively, such as by substitution, condensation, oxidation, reduction, attachment of a protecting group, and the like.

[0242] In this disclosure, the "active drug unit" is the portion of an antibody-drug conjugate (ADC) other than the antibody and linker, which is derived from the drug defined above. For convenience, the "active drug unit" in the ADC of this disclosure may be referred to directly by the name of the drug, as understood by those skilled in the art.

[0243] As used in this disclosure, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the antibody drug conjugate or drug linker conjugate of this disclosure, and the salt is not biologically or otherwise undesirable. The conjugates of this disclosure (including antibody drug conjugates and drug linker conjugates) can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In this disclosure, pharmaceutically acceptable acid addition salts represent salts formed between the conjugates of this disclosure and organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable base addition salts refer to salts of the conjugates disclosed herein formed with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium, or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing N groups. Pharmaceutically acceptable salts are preferred. However, other salts may also be useful, for example, in isolation or purification steps, and may be used during preparation, and are therefore encompassed within the scope of this application.

[0244] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0245] As used in this disclosure, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In a compound with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce a racemic mixture, a single enantiomer, a diastereomeric mixture, and a separate diastereomer. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of this disclosure can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this disclosure encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0246] Solid lines (-), solid wedges, or dashed wedges may be used in the present disclosure to depict carbon-carbon bonds of the compounds of the present disclosure. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom (e.g., specific enantiomers, racemic mixtures, etc.) are included. The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomers shown are present. Unless otherwise indicated, the compounds of the present disclosure are intended to exist in the form of stereoisomers (which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present disclosure may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereoisomer pairs).

[0247] The present disclosure also includes all pharmaceutically acceptable isotopic derivatives which are identical to the substances of the present disclosure except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the substances of the present disclosure include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H); carbon isotopes (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g.15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S). In some embodiments, the isotopic derivative is a deuterated derivative, ie, one in which one or more hydrogen atoms are deuterium atoms.

[0248] The compounds of the present disclosure may exist in the form of solvates (preferably hydrates), wherein the compounds of the present disclosure contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of the polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0249] Also included within the scope of the present disclosure are metabolites of the compounds of the present disclosure, i.e., substances formed in vivo upon administration of the compounds of the present disclosure. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. of the administered compound. Thus, the present disclosure includes metabolites of the compounds of the present disclosure, including compounds produced by contacting the compounds of the present disclosure with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0250] The present disclosure further includes within its scope prodrugs of the compounds of the present disclosure. Typically, such prodrugs will be functional group derivatives of the compounds that are easily converted into the desired therapeutically active compound in vivo. Therefore, in these cases, the term "administering" for the methods of treatment disclosed herein should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but after administration to an individual, the prodrug forms are converted into the above-mentioned compounds in vivo. For example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985, conventional methods for selecting and preparing suitable prodrug derivatives are described.

[0251] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These excipients are substances, other than active ingredients, that have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients or excipients also have important functions such as solubilization, dissolution assistance, and sustained-release control. They are important ingredients that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be categorized as natural, semi-synthetic, and fully synthetic. Based on their functions and uses, pharmaceutical excipients can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. Based on their route of administration, pharmaceutical excipients can be divided into oral, parenteral, mucosal, transdermal or topical, nasal or oral inhalation, and ocular administration. The same pharmaceutical excipient or vehicle can be used in pharmaceutical preparations for different routes of administration and have different functions and uses. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0252] The pharmaceutical composition can be made into various suitable dosage forms according to the route of administration. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye preparations, pills, implants, aerosols, powder sprays, sprays, etc. Wherein, the pharmaceutical composition or suitable dosage form can contain 0.01mg to 1000mg of the substance (including conjugates) of the present disclosure or a pharmaceutically acceptable salt thereof, preferably 0.1mg to 800mg, preferably 0.5-500mg, preferably 0.5 to 350mg, particularly preferably 1-250mg. Sometimes an amount exceeding the above range is also feasible.

[0253] The pharmaceutical composition can be administered in the form of an injection (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous injection), including injection solutions, sterile powders for injection, and concentrated solutions for injection. Among them, usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an infusion.

[0254] In the present disclosure, regarding “L is linked to the antibody via a sulfur atom,” those skilled in the art will understand that the sulfur atom comes from the sulfhydryl group contained in the antibody itself after the disulfide bond is opened (for example, reduction of the disulfide bond by the reducing agent TCEP can open the disulfide bond to generate a sulfhydryl group -SH). In other words, the -S- between L and Ab is not an additional external sulfur atom.

[0255] As used herein, the term "linker" or "linker" refers to a segment that connects an active drug unit (drug molecule) to an antibody portion. In this regard, the linker, prior to attachment to the antibody or antigen-binding fragment thereof (i.e., a precursor linker), has functional groups that can form bonds with functional groups of the antibody or antigen-binding fragment thereof.

[0256] In the present disclosure, the term "antibody drug conjugate" or "ADC" refers to a substance obtained by connecting an active drug unit (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the active drug unit is connected to the targeting portion via a linker. The linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound (e.g., a c-Myc protein degrader) fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the active drug unit is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the active drug unit from the antibody or its antigen-binding fragment.

[0257] The term "group of antibody drug conjugates" or "antibody drug conjugate population" refers to a group or mixture of the disclosed antibody drug conjugates, their tautomers, stereoisomers, isotopic derivatives (such as deuterated derivatives), prodrugs, or pharmaceutically acceptable salts or solvates, wherein the q of the antibody drug conjugates may be the same or different. Alternatively, it may be referred to as an "antibody drug conjugate mixture."

[0258] Advantageous Effects of the Invention

[0259] In some embodiments, the anti-VEGF antibody-drug conjugates disclosed herein utilize the extracellular cleavage ability of their linkers in the tumor microenvironment to bind to VEGF in the tumor microenvironment rather than to tumor cell surface antigens. The resulting antibody-drug conjugates still exhibit high anti-tumor activity. This overcomes the limitation of traditional ADC technology, which can only use antibodies that bind to tumor cell surface antigens, and develops ADCs with high anti-tumor activity targeting free VEGF.

[0260] In some embodiments, the antibody-drug conjugates (ADCs) disclosed herein have better solubility and / or excellent chemical stability. Experiments have shown that the VEGF-ADCs disclosed herein have excellent plasma stability and systemic circulation stability. The ADCs disclosed herein almost always prevent toxin shedding during incubation in plasma, thereby reducing the shedding of non-intended toxins in non-target cells and thus having a higher therapeutic index.

[0261] Through extensive research, this disclosure discovered a class of ADCs that can be cleaved in the tumor microenvironment (both inside and around tumor cells), enabling release of ADCs both inside and outside tumor cells via tissue proteases, maximizing the delivery of ADCs to tumor tissues and cells, thereby achieving tumor treatment efficacy. Therefore, anti-tumor effects can be achieved through free antigens such as VEGF.

[0262] In some embodiments, the antibody-drug conjugates disclosed herein utilize the extracellular cleavage ability of their linkers in the tumor microenvironment and their enrichment ability in the tumor microenvironment to form antibody-drug conjugates with antibodies that do not have the ability to endocytose and antibodies that do not have the ability to bind to tumor extracellular antigens. Such antibody-drug conjugates still have high anti-tumor activity.

[0263] The conjugate (ADC) obtained according to the above method increases the exposure of the entire ADC molecule in the relatively acidic tumor environment by adjusting the physicochemical properties of the linker and the entire ADC molecule. As a result, the ADC has better tumor tissue targeting, that is, the ability to be enriched in the tumor microenvironment, increases the ratio of the concentration of the bioactive molecule in the tumor and blood, and reduces the mechanism-related toxicity of the ADC molecule (toxicity caused by the ADC binding to cell surface antigens in non-tumor tissues, also known as "on-target toxicity"), thereby having a higher therapeutic index.

[0264] In some embodiments, the conjugate obtained according to the above method has high stability in the body circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of toxins in non-target tissues.

[0265] In some embodiments, the conjugate can synergistically exert the anti-tumor activity of the anti-VEGF antibody and the toxin, and its anti-tumor activity is better than that of the anti-VEGF antibody or the toxin alone or in combination.

[0266] In some embodiments, the bioactive molecule of the conjugate has higher anti-tumor cell activity and thus has an excellent bystander effect. The ADC can more effectively kill tumor cells with high antigen expression as well as tumor cells with low or no antigen expression in tumor tissues.

[0267] In summary, the ADC disclosed herein has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0268] Figure 1a. Species Cross-Reactivity of Anti-VEGF Antibody-Drug Conjugates

[0269] Figure 1b. Species cross-reactivity of anti-VEGF antibodies

[0270] Figure 2. Cross-reactivity of anti-VEGF antibody-drug conjugates with VEGF family proteins

[0271] Figure 3a. Statistical graph of the effect of anti-VEGF antibody-drug conjugate on HUVEC cell mesh formation

[0272] Figure 3b. Statistical graph of the effect of anti-VEGF antibody-drug conjugate on HUVEC cell intersection formation

[0273] Figure 3c. Fluorescence image of the effect of anti-VEGF antibody-drug conjugate on HUVEC cell tube formation

[0274] Figure 4. Efficacy test of anti-VEGF antibody-drug conjugate in SW480 transplanted tumor model

[0275] Figure 5. Effect of anti-VEGF antibody-drug conjugate on body weight in SW480 transplanted tumor model

[0276] Figure 6. Efficacy test of anti-VEGF antibody-drug conjugate in NCI-H358 transplanted tumor model

[0277] Figure 7. Effect of anti-VEGF antibody-drug conjugate on body weight in NCI-H358 transplanted tumor model

[0278] Figure 8. Anti-VEGF antibody-drug conjugate test on body weight of human colorectal cancer PDX model

[0279] Figure 9. Effect of anti-VEGF antibody-drug conjugate on body weight of human colorectal cancer PDX transplant tumor model DETAILED DESCRIPTION

[0280] The present disclosure is further described below by way of a description of specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or instruments used without indicating the manufacturer are conventional products that can be obtained commercially. Unless otherwise specified, the solutions mentioned in the examples are solutions in water. Unless otherwise specified, the abbreviations used herein generally have meanings known in the art.

[0281] Example 1. Preparation of anti-VEGF antibodies

[0282] Bevacizumab (trade name Avastin) is a VEGF inhibitor launched in the United States on February 26, 2004. It is used to treat various tumors, such as lung cancer and ovarian cancer. Its sequence and preparation method are disclosed in WO9845331. The sequences and preparation methods of this murine antibody and its humanized counterpart, rhuMab-VEGF (i.e., Bevacizumab), have also been disclosed in U.S. Patent No. 6,054,297 (inventors: Carter, Paul J. and Presta, Leonard G, patent application date: May 9, 1995, patent grant date: April 25, 2000, patent title: "Humanized antibodies and methods for making them") and published in Cancer Res (Presta LG et al., Cancer Res, 1997, 57:4593). The sequences and preparation methods of Bevacizumab or its biosimilars are also disclosed in CN109053895B.

[0283] Heavy chain sequence of Bevacizumab or its biosimilars:

[0284] Light chain sequence of Bevacizumab or its biosimilars:

[0285] The amino acid sequences of the CDR region, VH, and VL of Bevacizumab were obtained by analysis using techniques well known to those skilled in the art. The results are as follows:

[0286] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO: 3,

[0287] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 4,

[0288] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0289] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0290] An IgG1 isotype antibody was also prepared, which is a human anti-hen egg lysosomal antibody (human anti-Hen Egg Lysozyme IgG, anti-HEL, such as human IgG1, abbreviated as hIgG1). Its sequence was derived from the variable region sequence of the Fab F10.6.6 sequence in the study Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.). The preparation method is as follows: Nanjing GenScript Biotechnology was commissioned to perform amino acid codon optimization and gene synthesis on the heavy and light chain genes of the human IgG antibody. Referring to the standard techniques described in the Molecular Cloning Laboratory Manual (3rd Edition), the heavy and light chain genes were subcloned into the antibody heavy chain expression vector and antibody light chain expression vector of the mammalian expression system using standard molecular cloning techniques such as PCR, enzyme digestion, DNA gel recovery, ligation transformation, colony PCR or enzyme digestion identification, and the heavy and light chain genes of the recombinant expression vectors were further sequenced and analyzed. After sequencing verification, large quantities of endotoxin-free expression plasmids were prepared and transiently co-transfected into HEK293 cells with the heavy and light chain expression plasmids for recombinant antibody expression. After 7 days of culture, the cell culture fluid was harvested and affinity purified using an rProtein A column (GE). The harvested antibody sample was then analyzed using standard analytical techniques such as SDS-PAGE and SEC-HPLC for quality assurance.

[0291] Example 2: ADC preparation

[0292] Preparation of VEGF-ADC-01

[0293] q is selected from any integer between 1 and 10, and anti-VEGF mab is a monoclonal antibody that specifically binds to VEGF.

[0294] 2.1 VEGF-ADC-01 (DAR8) sample preparation

[0295] 1000 mg of the anti-VEGF antibody prepared in Example 1 was diluted with a diluent (20 mM phosphate (PB) buffer, pH 7.2), and a sodium edetate solution was added to a final concentration of 5 mM, mixed thoroughly. A 20 mmol / L TCEP solution (6.25 times the molar equivalent of the antibody) was added, mixed thoroughly, and allowed to stand at room temperature for 90 minutes. To this solution was added 10 mmol / L DL-037 (obtained according to Example 2.37 of WO2022170971, CAS No.: 2964513-44-6) dissolved in dimethyl sulfoxide (DMSO) (11 times the molar equivalent of the antibody), mixed thoroughly, and allowed to stand at room temperature for 2 hours to obtain a coupled sample. After completion of the reaction, the sample was replaced with a 20 mM histidine buffer (pH 6.0) using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-VEGF antibody ADC composition, VEGF-ADC-01 (DAR8). The average DAR value was determined by mass spectrometry as in Example 3 to be 8.1.

[0296] According to mass spectrometry, the antibody light chain on VEGF-ADC-01 (DAR8) is conjugated to 0-1 toxin molecules (obtained with reference to Example A1.9 of WO2022170971, CAS number 2821768-98-1) (the ratios of LC0 and LC1 are 0% and 100%, respectively), and the heavy chain is conjugated to 0-4 toxin molecules (the ratios of HC0, HC1, HC2, HC3, and HC4 are 0%, 0%, 0%, 97%, and 3%, respectively). It can be inferred that the q value can be 8, 9, or 10.

[0297] 2.2 VEGF-ADC-01 (DAR4) sample preparation

[0298] 1000 mg of the anti-VEGF antibody prepared in Example 1 was diluted with a diluent (20 mM PB, pH 7.2), and a sodium edetate solution was added to a final concentration of 5 mM and mixed. A 20 mmol / L TCEP solution (2.8 times the molar equivalent of the antibody) was added, mixed, and allowed to stand at room temperature for 90 minutes. To this solution was added 10 mmol / L DL-037 (obtained according to Example 2.37 of WO2022170971) dissolved in dimethyl sulfoxide (DMSO) at a molar equivalent of 4.8 times the antibody, mixed, and allowed to stand at room temperature for 2 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 20 mM histidine buffer at pH 6.0 using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-VEGF antibody ADC composition, VEGF-ADC-01 (DAR4). The average DAR value was determined by mass spectrometry to be 4.5.

[0299] Mass spectrometry analysis of VEGF-ADC-01 (DAR4) showed that the antibody light chain was conjugated to 0-1 toxin molecules (the ratios of LC0 and LC1 were 48% and 52%, respectively), and the heavy chain was conjugated to 0-4 toxin molecules (the ratios of HC0, HC1, HC2, HC3, and HC4 were 22%, 16%, 30%, 32%, and 0%, respectively). It can be inferred that the q value can be 1, 2, 3, 4, 5, 6, 7, or 8.

[0300] 2.3 Preparation of IgG1-ADC

[0301] 2.3.1 Preparation of IgG1-ADC (DAR8)

[0302] The antibody prepared in Example 2.1 was replaced with an IgG1 isotype antibody (anti-chicken lysozyme antibody, prepared in Example 1) to obtain the IgG1-ADC conjugated with DL-037. The DAR value was 8.0 as determined by mass spectrometry in Example 3.

[0303] 2.3.2 Preparation of IgG1-ADC (DAR4)

[0304] The antibody prepared in Example 2.2 was replaced with an IgG1 isotype antibody (anti-chicken lysozyme antibody, prepared in Example 1) to obtain the IgG1-ADC conjugated with DL-037. The DAR value was determined by mass spectrometry in Example 3 to be 4.4.

[0305] Example 3. Identification of Anti-VEGF ADC Preparation

[0306] Detection: LC-MS molecular weight and DAR value analysis was performed on VEGF-ADC-01 (DRA4) and VEGF-ADC-01 (DAR8). The chromatographic conditions were as follows:

[0307] Chromatographic column: PLRP-S, 2.1*50mm, 5μm;

[0308] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN

[0309] Column temperature: 70°C

[0310] Flow rate: 0.3ml / min

[0311] Injection volume: 1 μl

[0312] Sample treatment: Take 200 μg of sample, dilute to 0.2 mg / ml with ultrapure water, add 10 μl 1 mol / L DTT, react at 37°C for 30 min, and then inject directly.

[0313] LC / MS model: AB SCIEX X500B

[0314] Mass spectrometry conditions are as follows:

[0315] The result is as follows:

[0316] Table 1. Theoretical molecular weight and measured molecular weight

[0317] In Table 1, LC0 represents the unconjugated antibody light chain; LC1 represents the antibody light chain conjugated to one toxin; HC0 represents the unconjugated antibody heavy chain; HC1, HC2, HC3, and HC4 represent the antibody heavy chains conjugated to one, two, three, and four toxins, respectively. The theoretical molecular weights of LC0 and HC0 are calculated based on the amino acid sequence, while the theoretical molecular weight of HC is calculated based on the G0F glycoform desaturase.

[0318] Table 2. Proportions and DAR values ​​of each component

[0319] The test results showed that the drug-antibody coupling ratio (DAR value) of the VEGF-ADC-01 (DAR4) sample was 4.5; the drug-antibody coupling ratio (DAR value) of the VEGF-ADC-01 (DAR8) sample was 8.1.

[0320] Example 4, VEGF ADC protein affinity detection

[0321] The affinity of the anti-VEGF antibody prepared as described in Example 1 and the VEGF-ADC-01 (DAR4) prepared as described in Example 2 to various VEGF proteins was detected by ELISA. Specific experimental procedures: Human / monkey VEGF protein (VEGF165, P15692, from Sibowo), rat VEGF protein (VEGF164, purchased from Jinan Protein, product number CJ96) and mouse VEGF protein (VEGF164, purchased from Jinan Protein, product number CX73) were diluted with carbonate buffer with a pH value of 9.6 to a final concentration of 1 μg / mL, and 100 μL / well were added to 96-well enzyme-labeled wells and coated at 4°C overnight; the coating solution was discarded, and the wells were washed 3 times with PBST, and 300 μL PBS (containing 2% BSA) was added to each well and blocked at 37°C for 1 hour; then 100 μL was added to each well. Anti-VEGF antibody and VEGF-ADC-01 (DAR4) diluted in PBS (containing 2% BSA) (starting at 1000 ng / mL, four-fold dilutions, duplicate wells) were incubated at 37°C for 1.5 hours. The plates were washed three times with PBST. A horseradish peroxidase-conjugated anti-human Fab antibody (Invitrogen, A56868, diluted 1:2000 in PBS containing 2% BSA) was added at 100 μL / well for 1 hour at 37°C. The plates were then washed five times with PBST. TMB colorimetric solution was added for development (100 μL / well, 37°C, 7 minutes). The reaction was terminated with stop solution (50 μL / well), and absorbance was measured at 450 nm. EC50 values ​​were calculated based on the raw data. The results, as shown in Table 3 and Figures 1a and 1b, show that the anti-VEGF antibody and VEGF-ADC-01 (DAR4) strongly bound to human and monkey VEGF proteins, but not to rat and mouse VEGF proteins. Furthermore, the VEGF ADC's binding ability to VEGF antigens from different species was similar to that of the anti-VEGF antibody.

[0322] Table 3. Species cross-reactivity of anti-VEGF antibodies and VEGF ADCs

[0323] Example 5, VEGF ADC dynamic affinity detection

[0324] The dynamic affinity of the anti-VEGF antibody prepared as described in Example 1 and the VEGF-ADC-01 (DAR4) prepared as described in Example 2 for human VEGF protein was determined by surface plasmon resonance. The method is briefly described as follows: Human VEGF protein (Bepsix, VE5-H4210: isoform VEGF165) was desalted and replaced with HEPES buffer. After concentration determination using a SPECTROstar Nano (purchased from BMGLABTECH), serial dilutions were performed in HEPES buffer (starting at 62.5 nM, followed by two-fold dilutions for a total of seven concentrations). The anti-VEGF antibody and VEGF-ADC-01 (DAR4) were diluted to 6 μg / mL in HEPES buffer and injected into the experimental channel (Fc2) of a Protein A capture chip. Antigen dilutions were sequentially injected into the experimental channel (Fc2) and the reference channel (Fc1), with binding for 90 s and dissociation for 210 s in HEPES buffer. After one analysis cycle, the sensor was regenerated with 10 mM Gly (pH 1.5) for 30 seconds. Data were analyzed using Biacore Insight Evaluation Software using a 1:1 model. Association (Ka) and dissociation (Kd) rate constants were determined and used to calculate the dissociation equilibrium constant (KD). As shown in Table 4, the anti-VEGF antibody and VEGF-ADC-01 (DAR4) exhibited high dynamic affinity for human VEGF.

[0325] Table 4. Dynamic affinity analysis of anti-VEGF antibodies, VEGF ADCs, and VEGF protein

[0326] Example 6, VEGF ADC specificity detection

[0327] The binding properties of the anti-VEGF antibody (Bevacizumab) prepared as described in Example 1 and the VEGF-ADC-01 (DAR4) prepared as described in Example 2 to other VEGF family proteins were detected by ELISA experimental method. Specific experimental procedures: Human VEGFA protein (VEGF165, from Sibowo), human VEGF-B (purchased from Nearshore Protein, CS78), human VEGF-C (purchased from Nearshore Protein, C546), human VEGF-D (purchased from Nearshore Protein, C498), and human PLGF protein (purchased from Nearshore Protein, CW90) were diluted with carbonate buffer with a pH value of 9.6 to a final concentration of 1 μg / mL, and 100 μL / well were added to a 96-well ELISA plate and coated at 4°C overnight; the next day, the coating solution was discarded, the plate was washed 3 times with PBST, and 300 μL PBS (containing 2% The plates were blocked with BSA (BSA) at 37°C for 1.5 hours. After washing the plates three times with 1хPBST, the test sample (anti-VEGF antibody or VEGF-ADC-01 (DAR4)) was added at a concentration of 1000 ng / mL and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μL / well of horseradish peroxidase-conjugated anti-human Fab antibody (Invitrogen, A56868, diluted 1:2000 in PBS containing 2% BSA) was added and incubated at 37°C for 1 hour. The plates were then washed five times with PBST. TMB colorimetric solution (Huzhou Yingchuang, TMB-S-001) was added for color development. The reaction was terminated with stop solution and read on a microplate reader at 450 nm. The results are shown in Table 5 and Figure 2. The anti-VEGF antibody and VEGF-ADC-01 (DAR4) significantly bound to VEGFA but did not bind to VEGF-B, VEGF-C, VEGF-D, or PLGF.

[0328] Table 5. Specificity test results of anti-VEGF antibodies and VEGF ADC

[0329] Example 7: In vitro anti-angiogenesis assay of VEGF ADC

[0330] The effects of the anti-VEGF antibody prepared in Example 1 and the VEGF-ADC-01 (DAR4) prepared in Example 2 on the tube formation of vascular endothelial cells HUVEC (ATCC, catalog number CRL-1730) were detected by tube formation experiments.

[0331] Specific experimental operation: The extracellular matrix gel (Extracellular Matrix Gel) (BD, 354248) was placed at 4 ° C in advance to melt, and the 96-well plate and the tip of the gun were pre-cooled. 50 μL of matrix gel was added to each well and placed in a 37 ° C incubator for 30-45 minutes. The matrix gel was evenly solidified and spread on the bottom of the 96 wells. HUVEC cells in the logarithmic growth phase were added to a 96-well plate containing matrix gel at 10,000 per well, 50 μL / well, and then 3 μM / L anti-VEGF antibody (prepared in Example 1), 3 μM / L VEGF-ADC-01 (DAR4), 12 μM / L toxin (obtained with reference to Example A1.9 of WO2022170971), and 3.35uM / L Avastin (purchased from MCE, HY-P9906) were added to each experimental well to treat HUVEC cells. The experimental wells with only VEGF were used as controls. After 6 hours, the culture medium was washed off, and the cells were washed twice with 100 μL of washing solution. Each well was then stained with 100 μL of staining solution at 37°C for 0.5 h. Fluorescence images were taken under a 4x microscope. Indices of tube formation were calculated using Image J software and plotted using GraphPad Prism. The results are shown in Table 6 and Figures 3a-3c. Compared with the control group (VEGF), Avastin, toxins, anti-VEGF antibodies, and VEGF-ADC-01 (DAR4) significantly inhibited the formation of HUVEC cell networks and junctions, and reduced tube formation. In summary, toxins, anti-VEGF antibodies, and VEGF-ADC-01 (DAR4) significantly inhibited the ability of HUVEC cells to form tubes.

[0332] Table 6. Statistical results of HUVEC cell lumen formation

[0333] Example 8. Cellular Functional Activity of VEGF ADC

[0334] Because VEGF ADCs do not rely on specific cell endocytosis but are cleaved by enzymes in the tumor microenvironment to release toxins and exert their killing effects, this study used an in vitro model to simulate the cleavage process of VEGF ADCs by the tumor microenvironment and evaluate their cell-killing effects.

[0335] Specific experimental procedures: 200 μg / mL VEGF-ADC-01 (DAR4) was incubated with 50 μg / mL Cathepsin L (Biopsy, Catalog No. CAL-H5253) at 37°C for 72 hours to obtain the VEGF ADC enzyme incubation solution. NCI-H358 (human non-small cell lung cancer cells, sourced from ECACC, Catalog No. 95111733), SW480 (human colon cancer cells, sourced from ECACC, Catalog No. 87092801), SNU-16 (human gastric cancer cells, sourced from ATCC, Catalog No. CRL-5974), and NCI-H441 (human lung adenocarcinoma cells, sourced from ATCC, Catalog No. HTB-174) cells were cultured at 37°C in 5% CO2. Cells in the logarithmic growth phase were harvested and counted, and cell viability was determined by trypan blue staining to ensure that cell viability was above 90%. Adjust the cell density and seed 120 μL per well of a 96-well cell culture plate. For NCI-H358, SW480, SNU-16, and NCI-H441, seed 4,000, 6,000, 6,000, and 2,000 cells per well, respectively. Incubate the plates in an incubator overnight. The next day, prepare the sample solution. For each cell line, dilute the solution fourfold to a maximum working concentration of 260 nM. Dilutions are performed in triplicate for a total of nine concentrations. Add 30 μL / well of the sample solution to the 96-well plate. Incubate the 96-well plate with the samples in an incubator for an additional 6 days before analyzing cell viability. CellTiter Glo reagent (Promega, Cat. No. G7573) was thawed and the cell plate was equilibrated to room temperature for 30 minutes. 75 μL of CellTiter Glo reagent was added to each well and the cells were shaken on an orbital shaker for 2 minutes to lyse the cells. The cell plate was then left at room temperature for 10 minutes to stabilize the luminescence signal. Finally, the luminescence value was read and the data was collected. The results are shown in Table 7, demonstrating that the active ingredient released by the disclosed VEGF ADC after incubation with Cathepsin L has a significant killing effect on tumor cells.

[0336] Table 7. VEGF ADC enzyme incubation solution cell killing activity test results

[0337] NA means the IC50 value is too high and not available.

[0338] Example 9: Determination of in vivo activity of VEGF ADC

[0339] 9.1. Efficacy Testing of Anti-VEGF Antibody-Drug Conjugates on SW480 Tumor Transplants

[0340] The present disclosure demonstrates the in vivo efficacy of anti-VEGF antibodies and ADC drugs by evaluating their anti-tumor effects on a subcutaneous human colon cancer cell line SW480 cell (ATCC source) transplanted tumor model in Balb / c nude mice. The specific steps are as follows: 35 female Balb / c nude mice, 5-6 weeks old, were purchased from Chengdu Yaokang Biotechnology Co., Ltd. SW480 cells were cultured in a 15 cm diameter culture dish using 1640 medium containing 10% FBS. When the confluence reached about 80-90%, they were digested with trypsin-EDTA, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted using a cell counter and diluted with PBS to a cell concentration of 5×10 7 Balb / c nu mice were acclimated to the laboratory environment for 3-5 days and then inoculated subcutaneously with SW480 cells in the right rib cage. The inoculated cell volume was 5×10 6 / mouse, the inoculation volume was 0.2 ml (containing 50% Matrigel), and the tumor was grown to 180 mm 3 Around 6:00 p.m., mice were randomly divided into groups based on tumor size, with five mice per group: a saline group (vehicle), an anti-VEGF antibody group, an anti-chicken lysozyme human IgG1-ADC (DAR8) (prepared in Example 2), a VEGF-ADC-01 (DAR4) dose group, and a VEGF-ADC-01 (DAR8) dose group. All samples were injected into the tail vein once a week for a total of 3 weeks. After administration, the mice were observed and their tumor volume and body weight were measured regularly.

[0341] 1) Relative tumor growth rate, T / C (%), that is, the relative tumor volume or tumor weight percentage between the treatment group and the control group at a certain time point.

[0342] 2) Relative tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment and control groups at a specific time point, respectively).

[0343] Specific results are shown in Table 8 and Figure 4. The experimental results show that all dose groups inhibited tumor growth in the SW480 human colon cancer xenograft model. VEGF-ADC-01(DAR4) and VEGF-ADC-01(DAR8) demonstrated significant tumor growth inhibition across all dose groups, with a dose-dependent trend. Body weight results are shown in Figure 5, demonstrating a favorable safety profile.

[0344] Table 8. SW480+Balb / c Nude mouse model efficacy results *: Due to the rapid growth of the tumor in the control group, the tumor was dissected in advance according to animal ethics and welfare requirements. The volume is the tumor volume on the day of dissection (D19). **: Dunnett's test was used to perform statistical analysis between each treatment group and the normal saline control group. Compared with the IgG1-ADC (DAR8) group, ## Indicates p<0.01, ### p < 0.001, and p < 0.05 was considered to be significantly different.

[0345] 9.2. Efficacy Testing of Anti-VEGF Antibody-Drug Conjugates on NCI-H358 Tumor Xenografts

[0346] The present disclosure demonstrates the in vivo efficacy of anti-VEGF antibodies and ADC drugs by evaluating the anti-tumor effects on the subcutaneous human non-small cell lung cancer cell line NCI-H358 cell (Nanjing Kebai Biotechnology) transplanted tumor model in Balb / c Nude mice. The specific steps are as follows: 56 Balb / c nude mice, female, 5-6 weeks old, were purchased from Chengdu Yaokang Biotechnology Co., Ltd. NCI-H358 cells were cultured in 15 cm diameter culture dishes with 1640 medium containing 10% FBS, digested with trypsin-EDTA when the confluence reached about 80-90%, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted using a cell counter and diluted with PBS to a cell concentration of 5×10 7 Balb / c nu mice were acclimated to the laboratory environment for 3-5 days and NCI-H358 cells were inoculated subcutaneously in the right rib cage at a volume of 5×10 6 / mouse, the inoculation volume was 0.2 ml (containing 50% Matrigel), and the tumor was grown to 180 mm 3 At about 14 days, the mice were randomly divided into groups according to tumor size, with 5 mice in each group, namely, normal saline group, anti-VEGF antibody group, toxin group (obtained with reference to Example A1.9 of WO2022170971), anti-chicken lysozyme human IgG1-ADC (DAR4) (prepared in Example 2), and VEGF-ADC-01 (DAR4) 0.5 mg / kg, 1 mg / kg, and 3 mg / kg dose groups. All samples were injected into the tail vein once a week for a total of 3 weeks. After administration, the tumor volume and body weight of the mice were observed and measured regularly.

[0347] 1) Relative tumor growth rate, T / C (%), that is, the relative tumor volume or tumor weight percentage between the treatment group and the control group at a certain time point.

[0348] 2) Relative tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment and control groups at a specific time point, respectively).

[0349] Detailed results are shown in Table 9 and Figure 6. The experimental results demonstrate that all dose groups of VEGF-ADC-01 (DAR4) significantly inhibited tumor growth in the NCI-H358 human non-small cell lung cancer xenograft model, with a dose-dependent trend. Body weight results are shown in Figure 7, demonstrating a favorable safety profile.

[0350] Table 9. Efficacy results of NCI-H358 Balb / c Nude mouse model *: Dunnett's test was used to analyze the statistical data of each drug-treated group and the normal saline control group. ## p < 0.01, and p < 0.05 was considered to be significantly different.

[0351] 9.3. Efficacy Testing of Anti-VEGF Antibody-Drug Conjugates on LD1-2012-200671 PDX Tumor Xenografts

[0352] The present disclosure demonstrates the in vivo efficacy by evaluating the anti-tumor effects of anti-VEGF antibodies and ADC drugs on the subcutaneous human xenograft tumor (PDX) model of human colorectal cancer LD1-2012-200671 (Shanghai Lidi Biotechnology Co., Ltd.). The specific steps are as follows: 16 female NU / NU mice were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. The tumor tissue of human colorectal cancer LD1-2012-200671 was evenly cut into tumor pieces of about 3mm×3mm×3mm in size and weighing about 30-60mg and inoculated into the right side of the NU / NU mouse subcutaneously. When the tumor grew to 157mm 3 Around 6:00 p.m., mice were randomly divided into groups of four based on tumor size: a saline group (vehicle), an anti-chicken lysozyme human IgG1-ADC (DAR4) (prepared in Example 2), an anti-VEGF antibody group, and a VEGF-ADC-01 (DAR4) dose group. All samples were injected into the tail vein once weekly for three weeks. Following administration, the mice were observed and their tumor volume and body weight were measured regularly.

[0353] 1) Relative tumor growth rate, T / C (%), that is, the relative tumor volume or tumor weight percentage between the treatment group and the control group at a certain time point.

[0354] 2) Relative tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment and control groups at a specific time point, respectively).

[0355] Specific results are shown in Table 10 and Figure 8. The experimental results show that all ADC dose groups were able to inhibit tumor growth in the LD1-2012-200671 mouse subcutaneous colon cancer PDX model, and VEGF-ADC-01 (DAR4) demonstrated superior inhibitory efficacy compared to IgG1-ADC (DAR4) and anti-VEGF antibodies. Body weight results are shown in Figure 9, demonstrating a favorable safety profile.

[0356] Table 10. LD1-2012-200671 PDX mouse model efficacy results **: One-Way ANOVA Uncorrected Fisher's LSD test was used to perform statistical analysis on each drug-treated group and the normal saline control group. ## p < 0.01, and p < 0.05 was considered to be significantly different.

[0357] Example 10: ADC plasma stability test

[0358] Solution preparation: Dilute the ADC stock solution to be tested with PBS to a working solution with a concentration of 2 mg / mL.

[0359] Sample incubation: The antibacterial agent ProClin was added to the VEGF-ADC-01 working solution and human plasma, respectively, to a final concentration of 0.1%. The antibacterial agent-added ADC solution was added to the sterile plasma at a final ADC concentration of 200 μg / ml. The cells were incubated in a 37°C cell culture incubator at 80 oscillations / minute. After incubation for 0, 7, and 14 days, ADC samples were removed and 100 μl of Protein A was added to each tube. The adsorption was allowed to proceed for 2 hours, and the cells were eluted to obtain the post-incubation ADCs. The DAR values ​​of the post-incubation ADCs were measured (as in Example 3) to determine the plasma stability of the samples.

[0360] The test results show that the DAR values ​​of VEGF-ADC-01 (DAR4) and VEGF-ADC-01 (DAR8) disclosed herein remain basically stable during plasma incubation (i.e., almost no toxin linker falls off from the antibody), and have good plasma stability.

[0361] Example 11, ADC toxicity test

[0362] The present invention discloses administering different doses of VEGF-ADC-01 (DAR4) to cynomolgus monkeys by intravenous infusion, repeating the administration for 4 weeks, stopping the drug for 4 weeks, observing the toxic reactions and their reversibility, and determining the target organs or target tissues of the toxicity. The specific steps are as follows: select cynomolgus monkeys, half male and half female, and administer a vehicle (glucose solution) control or different doses of VEGF-ADC-01 (DAR4) by intravenous infusion, administering once every 2 weeks for a total of 3 doses, and stopping the drug for 4 weeks. All surviving animals were euthanized and dissected at the end of the dosing period and the end of the recovery period. Evaluation indicators include clinical observation, body weight, food intake, body temperature, ophthalmological examination, clinical pathology, gross anatomical macroscopic observation, organ weight and histopathological examination.

[0363] The results showed that under the experimental conditions, the highest non-serious toxicity dose (HNSTD) of VEGF-ADC-01 (DAR4) in crab-eating macaques was 30 mg / kg. No serious toxic reactions were observed in this experiment, only mild toxic reactions, which were all recovered after drug discontinuation.

[0364] Although the specific embodiments of the present disclosure have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings published, and that such modifications are within the scope of protection of the present disclosure. The scope of protection of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate, which is a compound of formula (I), or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative) or a pharmaceutically acceptable salt or solvate thereof: Ab-[LD]q Formula (I) in, Ab is an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF); L is a bond or linking molecule that covalently links Ab and D; q is an integer between 1 and 16 and represents the number of LDs covalently linked to Ab; preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; D is a camptothecin drug molecule.

2. The antibody-drug conjugate or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate according to claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to VEGF comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein: The CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact definition schemes, or any combination thereof; Optionally, the antibody or antigen-binding fragment thereof that specifically binds to VEGF comprises: Three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Chothia scheme, Three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the AbM scheme, Three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Kabat scheme, three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the IMGT scheme, or The three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and the three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Contact scheme; Preferably, the antibody or antigen-binding fragment thereof that specifically binds to VEGF comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 in the Chothia, AbM, Kabat, IMGT, or Contact definition scheme, wherein (i) According to the Chothia scheme definition, CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:5; CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 10; CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23; CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 22; (ii) according to the AbM protocol definition, CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:6; CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 11; CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23; CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 22 (iii) according to the Kabat protocol definition, CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:7; CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 12; CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 23; CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 22; (iv) According to the Contact scheme definition, CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:8; CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 13; CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 15; CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:24; CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 25; and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:21; (v) According to the IMGT scheme definition, CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:9; CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 14; CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 16; CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 20; CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 18; and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 22; (vi) According to the Chothia scheme definition, CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:5; CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 10; CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23; CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22; (vii). According to the AbM protocol definition, CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:6; CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 11; CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23; CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 consists of the amino acid sequence shown in SEQ ID NO: 22 (viii) According to the Kabat protocol definition, CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:7; CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 12; CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 17; CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:23; CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 19; and CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:22; (ix). According to the Contact solution definition, CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:8; CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 13; CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 15; CDR-L1 consists of the amino acid sequence shown in SEQ ID NO:24; CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 25; and CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:21; (x). According to the IMGT scheme definition, CDR-H1 consists of the amino acid sequence shown in SEQ ID NO:9; CDR-H2 consists of the amino acid sequence shown in SEQ ID NO: 14; CDR-H3 consists of the amino acid sequence shown in SEQ ID NO: 16; CDR-L1 consists of the amino acid sequence shown in SEQ ID NO: 20; CDR-L2 consists of the amino acid sequence shown in SEQ ID NO: 18; and CDR-L3 consists of the amino acid sequence shown in SEQ ID NO:

22.

3. The antibody-drug conjugate according to claim 1 or 2, or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate, wherein Ab comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; or (ii) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 4; (iii) the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:4; or (iv) the heavy chain variable region comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

4.

4. The antibody-drug conjugate according to any one of claims 1 to 3, or its tautomer, stereoisomer, isotope derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate, wherein Ab comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 2; (ii) the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain consists of the amino acid sequence shown in SEQ ID NO: 2; (iii) the heavy chain comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that has 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2; or (iv) the heavy chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2; Preferably, Ab is selected from Bevacizumab or a biosimilar thereof or an antigen-binding fragment thereof; Preferably, Ab is selected from any of the following Bevacizumab biosimilars or antigen-binding fragments thereof: ABP 215, BCD-021, FKB238, Mvasi (Bevacizumab-awwb), PF-06439535, SB8, Zirabev (Bevacizumab-bvzr), QL1101, IBI305, SHR-1603, BAT1706, HS008, MIL60 or an antigen-binding fragment thereof.

5. The antibody-drug conjugate according to any one of claims 1 to 4, or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or a pharmaceutically acceptable salt or solvate thereof, wherein L comprises an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues; Preferably, L comprises an amino acid residue that can be cleaved in the tumor microenvironment or a short peptide consisting of 2-10 amino acid residues; Optionally, the amino acid residue in L is a natural amino acid residue or a modification thereof; Preferably, L is selected from Among them, position 1 is connected to Ab and position 2 is connected to D.

6. The antibody-drug conjugate according to any one of claims 1 to 5, or a tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or a pharmaceutically acceptable salt or solvate thereof, wherein D is selected from camptothecin, SN38, DXD, a substituent-modified camptothecin, a substituent-modified DXD, or a substituent-modified SN38; Alternatively, D is selected from: One of them is connected to L.

7. The antibody-drug conjugate according to any one of claims 1 to 6, or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate, wherein the compound of formula (I) is selected from: in, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the antibody or antigen-binding fragment thereof that specifically binds to VEGF comprises: three CDR-Hs of the heavy chain variable region (VH) sequence of SEQ ID NO: 3 and three CDR-Ls of the light chain variable region (VL) sequence of SEQ ID NO: 4, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact schemes or any combination thereof; More preferably, the antibody or antigen-binding fragment thereof Ab that specifically binds to VEGF comprises: a heavy chain variable region (VH) of SEQ ID NO: 3 and a light chain variable region (VL) of SEQ ID NO:

4. Further preferably, Ab is selected from Bevacizumab or an antigen-binding fragment thereof, or a biosimilar thereof.

8. A group of antibody drug conjugates comprising or consisting of the antibody-drug conjugate according to any one of claims 1 to 7 or its tautomers, stereoisomers, isotopic derivatives (such as deuterated derivatives) or pharmaceutically acceptable salts or solvates, wherein The antibody drug conjugate has one, two or more q values; Optionally, when the antibody drug conjugates in the antibody drug conjugate population have two or more q values, the proportion of antibody drug conjugates with a specific q value in all antibody drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%; Optionally, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5; Preferably, the average drug to antibody ratio (average DAR) of the population of antibody drug conjugates is selected from about 2.0, 4.0, 6.0 or 8.

0.

9. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 7, or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate, or the antibody-drug conjugate group according to claim 8, and optionally one or more pharmaceutical excipients, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer; Optionally, the drug-to-antibody ratio (average DAR) in the pharmaceutical composition or antibody-drug conjugate population is an integer or decimal selected from 1-10; Preferably, the drug to antibody ratio (average DAR) in the pharmaceutical composition or antibody drug conjugate population is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5 and 7.5-8.5; Preferably, the DAR in the pharmaceutical composition or antibody drug conjugate population is selected from the group consisting of: 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, and 8±0.5; Preferably, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody drug conjugates is selected from the group consisting of about 2.0, 4.0, 6.0 and 8.

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10. Use of the antibody-drug conjugate according to any one of claims 1 to 7, or its tautomer, stereoisomer, isotopic derivative (such as a deuterated derivative), or pharmaceutically acceptable salt or solvate, the antibody-drug conjugate group according to claim 8, or the pharmaceutical composition according to claim 9, as a medicament or for the preparation of a medicament for treating or preventing a disease related to the activity of VEGF or a disease related to a target of VEGF, including tumors, such as cancers, such as solid tumors or hematological tumors; Optionally, the tumor is selected from lung cancer (eg, non-small cell lung cancer, lung adenocarcinoma), colon cancer (eg, human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (eg, colorectal adenocarcinoma).