Anti-met / EGFR bispecific antibody and drug conjugate thereof

Abstract

Provided is an anti-MET nanobody and an anti-EGFR nanobody, and a bispecific antibody comprising the two nanobodies. Also provided is a drug conjugate constructed on the basis of the nanobodies and the bispecific nanobody.

Description

An anti-MET / EGFR bispecific antibody and drug conjugate thereof Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to an anti-MET / EGFR bispecific antibody and a drug conjugate thereof. Background Art

[0002] ADC (antibody-drug conjugate) technology is a current research hotspot in cancer treatment. It uses monoclonal antibodies to specifically bind to their targets, carry small molecule drugs to lesions, and release the drugs to exert their efficacy, thereby killing tumor cells and treating cancer. Because tumorigenesis is multifactorial, single-target therapies can easily lead to drug resistance and damage normal tissue cells. Therefore, single-target ADCs cannot fully meet the needs of cancer treatment. There is an urgent need to develop dual-target ADC drugs to reduce cytotoxicity to normal tissues and alleviate drug resistance.

[0003] MET (Mesenchymal-Epithelial Transition Factor) and EGFR (Epidermal Growth Factor Receptor) are classic targets for cancer treatment. Both are receptor tyrosine kinase proteins closely associated with pathological activities such as tumor development, metastasis, and drug resistance. MET amplification is one of the factors contributing to tumor cell resistance to EGFR inhibitors. For example, in metastatic colorectal cancer resistant to cetuximab or panitumumab, MET gene amplification occurs. Inhibiting MET can effectively suppress tumor growth in cetuximab-resistant PDX mouse models. Given the association between EGFR and MET in tumor development, simultaneous inhibition of MET and EGFR has become a strategy for anti-tumor drug development.

[0004] Almost all ADCs in the prior art use traditional antibodies such as IgG1 and IgG4 coupled to toxins. The currently available MET-EGFR dual-target antibody is Amivantamab, which is used to treat non-small cell lung cancer with EGFR exon 20 insertion mutations. AstraZeneca announced the MET-EGFR dual-target ADC patent number (US20230183358A1) of AZD9592, which is currently in the clinical phase 1 research stage. In addition, the MET-EGFR ADC in Sorrento's WO2018069851A2 patent is in the preclinical research stage. Although traditional antibodies are widely used and have good targeting, they have a large molecular weight, limited tumor penetration ability, and slow distribution. Compared with traditional antibodies, nanoantibodies are composed only of heavy chain dimers, including VHH region, CH2 region, and CH3 region, with a smaller molecular weight. In theory, they have a stronger diffusion ability in tumor tissue, which is more conducive to ADC drugs entering cells and releasing toxins.

[0005] In addition, the prior art also has the following defects:

[0006] 1) Low product purity: The drug-antibody ratio (DAR) of currently available ADC drugs is mainly distributed in the range of 3-5, such as Tisotumab Vedotin-tftv, Brentuximab Vedotin, Disitamab Vedotin, Enfortumab Vedotin-ejfv, etc. The above ADCs all use the cysteine ​​method to couple antibodies and toxins. Since the antibodies used are all traditional IgG1 antibodies, this type of antibody has four pairs of disulfide bonds in the hinge region and can be coupled to up to 8 toxins through the cysteine ​​coupling method, the coupling mixture contains coupling products of DAR2, DAR4, DAR6, and DAR8. The process is difficult to control and the product is complex. For example, in AbbVie's US20150337042 patent, the ADC product targeting EGFR has 8 HIC peaks, mainly distributed at the DAR2 and DAR4 peaks. The purity of the DAR4 product is 35.28%, which reaches 45.88% after purification; the DAR4 product of ABBV-399 in AU2017268342B2 only accounts for 26%.

[0007] 2) Toxic Side Effects: EGFR is widely expressed in normal tissues, with particularly high expression in the skin and thyroid. Therefore, EGFR inhibition can easily cause normal tissue damage. Current development of EGFR antibodies and ADCs focuses on reducing affinity, using affinity-reduced EGFR antibodies for toxin conjugation to minimize toxicity to normal tissues. However, excessively low affinity can compromise cytotoxicity, and achieving this balance requires considerable exploration.

[0008] Therefore, it is necessary to develop MET-EGFR dual-target ADC drugs based on nanobodies with smaller molecular weight in this field.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide an anti-MET / EGFR bispecific antibody and a drug conjugate thereof.

[0011] In a first aspect of the present invention, a Nanobody targeting MET is provided, wherein the VHH chain of the Nanobody has the following complementarity determining regions CDR1, CDR2 and CDR3:

[0012] CDR1 shown in SEQ ID NO: 13,

[0013] CDR2 shown in SEQ ID NO: 14, and

[0014] CDR3 shown in SEQ ID NO:15.

[0015] In another preferred example, the CDR region of the Nanobody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0016] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain MET binding affinity.

[0017] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, and more preferably 1.

[0018] In another preferred embodiment, the VHH chain of the Nanobody further comprises a framework region (FR).

[0019] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0020] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0021] In another preferred embodiment, the Nanobody binds to human, mouse or monkey MET.

[0022] In another preferred embodiment, the nanobody can be internalized by cells expressing the MET antigen.

[0023] In another preferred embodiment, the Nanobody blocks the binding between MET and HGF.

[0024] In another preferred example, the VHH chain of the Nanobody targeting MET has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homologous to the amino acid sequence shown in SEQ ID NO: 1.

[0025] In another preferred example, the VHH chain of the MET-targeting Nanobody has the amino acid sequence shown in SEQ ID NO: 1.

[0026] In the second aspect of the present invention, an antibody targeting MET is provided, wherein the antibody comprises one or more VHH chains of the Nanobody targeting MET as described in the first aspect of the present invention.

[0027] In another preferred example, the VHH chain of the MET-targeting Nanobody has the amino acid sequence shown in SEQ ID NO: 1.

[0028] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0029] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0030] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0031] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0032] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0033] In another preferred embodiment, the antibody is a monoclonal antibody.

[0034] In another preferred embodiment, the antibody is a partially or fully humanized antibody.

[0035] In another preferred embodiment, the antibody is a heavy chain antibody, and the heavy chain antibody comprises heavy chain constant regions CH2 and CH3 (Fc segment).

[0036] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0037] In another preferred example, the amino acid sequence of the heavy chain antibody is shown in SEQ ID NO: 2.

[0038] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain MET binding affinity.

[0039] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.

[0040] In another preferred embodiment, the antibody binds to human, mouse or monkey MET.

[0041] In another preferred embodiment, the antibody can be internalized by cells expressing MET antigen.

[0042] In another preferred embodiment, the antibody blocks the binding between MET and HGF.

[0043] In a third aspect of the present invention, there is provided a Nanobody targeting EGFR, the VHH chain of said Nanobody having the following complementarity determining regions CDR1, CDR2 and CDR3:

[0044] CDR1 shown in SEQ ID NO: 16,

[0045] CDR2 shown in SEQ ID NO: 17, and

[0046] CDR3 shown in SEQ ID NO:18.

[0047] In another preferred example, the CDR region of the Nanobody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0048] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain EGFR binding affinity.

[0049] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, and more preferably 1.

[0050] In another preferred embodiment, the VHH chain of the Nanobody further comprises a framework region (FR).

[0051] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0052] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0053] In another preferred embodiment, the nanobody binds to human, mouse or monkey EGFR.

[0054] In another preferred embodiment, the nanobody can be internalized by cells expressing EGFR antigen.

[0055] In another preferred embodiment, the Nanobody does not block the binding of EGFR and EGF.

[0056] In another preferred example, the VHH chain of the Nanobody targeting EGFR has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homologous to the amino acid sequence shown in SEQ ID NO: 3.

[0057] In another preferred example, the VHH chain of the Nanobody targeting EGFR has the amino acid sequence shown in SEQ ID NO: 3.

[0058] In the fourth aspect of the present invention, an antibody targeting EGFR is provided, wherein the antibody comprises one or more VHH chains of the Nanobody targeting EGFR as described in the third aspect of the present invention.

[0059] In another preferred example, the VHH chain of the Nanobody targeting EGFR has the amino acid sequence shown in SEQ ID NO: 3.

[0060] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0061] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0062] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0063] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0064] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0065] In another preferred embodiment, the antibody is a monoclonal antibody.

[0066] In another preferred embodiment, the antibody is a partially or fully humanized antibody.

[0067] In another preferred embodiment, the antibody is a heavy chain antibody, and the heavy chain antibody comprises heavy chain constant regions CH2 and CH3 (Fc segment).

[0068] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0069] In another preferred example, the amino acid sequence of the heavy chain antibody is shown in SEQ ID NO: 4.

[0070] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain EGFR binding affinity.

[0071] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.

[0072] In another preferred embodiment, the antibody binds to human, mouse or monkey EGFR.

[0073] In another preferred embodiment, the antibody can be internalized by cells expressing EGFR antigen.

[0074] In another preferred embodiment, the antibody does not block the binding between EGFR and EGF.

[0075] In the fifth aspect of the present invention, a multispecific antibody is provided, which comprises: the VHH chain of the Nanobody targeting MET as described in the first aspect of the present invention, the antibody targeting MET as described in the second aspect of the present invention, the VHH chain of the Nanobody targeting EGFR as described in the third aspect of the present invention, the antibody targeting EGFR as described in the fourth aspect of the present invention, or a combination thereof.

[0076] In another preferred embodiment, the multispecific antibody is a bispecific antibody targeting MET and EGFR.

[0077] In another preferred embodiment, the multispecific antibody comprises one or more antigen-binding chains, each of which independently has a structure represented by formula (I) from N-terminus to C-terminus: V1-L1-V2-H1-Fc1-L2-V3 (I)

[0078] in,

[0079] V1, V2, and V3 are each independently none, a VHH chain of the Nanobody targeting MET, or a VHH chain of the Nanobody targeting EGFR, and at least one of V1, V2, and V3 is a VHH chain of a Nanobody targeting MET or EGFR;

[0080] L1 and L2 are each independently none or a linker peptide;

[0081] H1 is the absence or immunoglobulin hinge region;

[0082] Fc1 is the immunoglobulin Fc segment;

[0083] “-” represents a peptide bond.

[0084] In another preferred embodiment, the bispecific antibody is a dimer consisting of a first antigen-binding chain and a second antigen-binding chain.

[0085] In another preferred embodiment, the bispecific antibody is a homodimer or a heterodimer.

[0086] In another preferred embodiment, the bispecific antibody has a structure shown in Formula II from N-terminus to C-terminus:

[0087] in,

[0088] V1, V2, V3, V4, V5, and V6 are each independently none, a VHH chain of the Nanobody targeting MET, or a VHH chain of the Nanobody targeting EGFR,

[0089] At least one of V1, V2, and V3 is a VHH chain of a nanobody targeting MET, and at least one of V4, V5, and V6 is a VHH chain of a nanobody targeting EGFR;

[0090] L1, L2, L3, L4, L5, and L6 are each independently none or a connecting peptide;

[0091] H1 and H2 are each independently free or an immunoglobulin hinge region;

[0092] Fc1 and Fc2 are each independently an immunoglobulin Fc segment;

[0093] “‖” represents a disulfide bond or a knob-into-hole connection;

[0094] “-” represents a peptide bond.

[0095] In another preferred embodiment, the connecting peptide is a flexible connecting peptide.

[0096] In another preferred embodiment, the connecting peptide is represented by (G4S)n, wherein n is an integer selected from 1-6, preferably 2, 3, 4 or 5.

[0097] In another preferred embodiment, the Fc1 and / or Fc2 are derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0098] In another preferred embodiment, the H1 and / or H2 are derived from the hinge region of IgG, preferably from the hinge region of human IgG.

[0099] In another preferred embodiment, the H1 and / or H2 are truncated at the N-terminus by 1-7 amino acids, such as 3, 4 or 5 amino acids, compared to the hinge region of a wild-type immunoglobulin (such as human IgG).

[0100] In another preferred embodiment, the sequences of H1 and H2 are independently shown as SEQ ID NO: 23 or SEQ ID NO: 24.

[0101] In another preferred embodiment, the sequence of H2 is shown in SEQ ID NO: 24.

[0102] In another preferred embodiment, the bispecific antibody has a structure represented by the following formula (IIa) from N-terminus to C-terminus:

[0103] in,

[0104] One of V1 and V2 is the VHH chain of a nanobody targeting MET, and the other is the VHH chain of a nanobody without or targeting EGFR;

[0105] One of V4 and V5 is the VHH chain of a Nanobody targeting EGFR, and the other is the VHH chain of a Nanobody with or without MET;

[0106] The remaining elements are as defined in formula (II).

[0107] In another preferred embodiment, the V1 is the VHH chain of a nanobody targeting MET, and V2 is absent.

[0108] In another preferred embodiment, the V1 is the VHH chain of a nanobody targeting MET, and V2 is the VHH chain of a nanobody targeting EGFR.

[0109] In another preferred embodiment, the V4 is the VHH chain of a nanobody targeting EGFR, and V5 is the VHH chain of a nanobody targeting MET.

[0110] In another preferred embodiment, the V4 is the VHH chain of a nanobody targeting MET, and V5 is the VHH chain of a nanobody targeting EGFR.

[0111] In another preferred embodiment, the bispecific antibody has a structure represented by the following formula (IIb) from N-terminus to C-terminus:

[0112] in,

[0113] V1 is the VHH chain of the nanobody targeting MET;

[0114] V4 is the VHH chain of the nanobody targeting EGFR;

[0115] The remaining elements are as defined in formula (II).

[0116] In another preferred embodiment, the bispecific antibody has a structure represented by the following formula (IIc) from N-terminus to C-terminus:

[0117] in,

[0118] V1 and V4 are each independently a VHH chain of a Nanobody targeting MET;

[0119] V3 and V6 are each independently a VHH chain of a Nanobody without or targeting EGFR, and at least one of V3 and V6 is a VHH chain of a Nanobody targeting EGFR;

[0120] The remaining elements are as defined in formula (II).

[0121] In another preferred embodiment, the amino acid sequence of the antigen-binding chain is selected from the following group:

[0122] (a) the amino acid sequence shown in any one of SEQ ID NOs: 2, 5-12;

[0123] (b) A derivative sequence formed by adding one or more amino acids, substituting one or more amino acids, or deleting 1-30 amino acids to the amino acid sequence in (a), wherein the derivative sequence retains the antigen-specific binding ability of its source sequence.

[0124] In another preferred embodiment, the amino acid sequence of the first antigen-binding chain is selected from the following group:

[0125] (a) the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 11, and 12;

[0126] (b) A derivative sequence formed by adding one or more amino acids, substituting one or more amino acids, or deleting 1-30 amino acids to the amino acid sequence in (a), wherein the derivative sequence retains the antigen-specific binding ability of its source sequence.

[0127] In another preferred embodiment, the amino acid sequence of the second antigen-binding chain is selected from the following group:

[0128] (a) the amino acid sequence shown in any one of SEQ ID NOs: 5, 7-12;

[0129] (b) A derivative sequence formed by adding one or more amino acids, substituting one or more amino acids, or deleting 1-30 amino acids to the amino acid sequence in (a), wherein the derivative sequence retains the antigen-specific binding ability of its source sequence.

[0130] In another preferred embodiment, the bispecific antibody comprises the first antigen-binding chain shown in SEQ ID NO: 2 and / or the second antigen-binding chain shown in SEQ ID NO: 5 or 7.

[0131] In another preferred embodiment, the multispecific antibody further comprises an antigen binding region targeting other antigens, and preferably, the antigen binding region is a nanobody.

[0132] In another preferred embodiment, the affinity KD value of the multispecific antibody for human MET is ≤10-8 M, preferably ≤10 -9 M.

[0133] In another preferred embodiment, the affinity KD value of the multispecific antibody to human EGFR is ≤10 -5 M, preferably ≤10 -6 M.

[0134] In a sixth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0135] (i) a Nanobody targeting MET as described in the first aspect of the invention, an antibody targeting MET as described in the second aspect of the invention, a Nanobody targeting EGFR as described in the third aspect of the invention, an antibody targeting EGFR as described in the fourth aspect of the invention, or a multispecific antibody as described in the fifth aspect of the invention; and

[0136] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0137] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0138] In the seventh aspect of the present invention, a chimeric antigen receptor (CAR) construct is provided, the antigen binding region of the CAR construct comprising a nanobody targeting MET as described in the first aspect of the present invention, a nanobody targeting EGFR as described in the third aspect of the present invention, or a combination thereof.

[0139] In the eighth aspect of the present invention, a recombinant immune cell is provided, wherein the immune cell expresses an exogenous CAR construct as described in the seventh aspect of the present invention.

[0140] In another preferred embodiment, the immune cells are selected from NK cells or T cells.

[0141] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0142] In a ninth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0143] (a) an antibody portion, which is a Nanobody targeting MET as described in the first aspect of the invention, an antibody targeting MET as described in the second aspect of the invention, a Nanobody targeting EGFR as described in the third aspect of the invention, an antibody targeting EGFR as described in the fourth aspect of the invention, or a multispecific antibody as described in the fifth aspect of the invention; and

[0144] (b) a coupling moiety coupled to said Nanobody portion, said coupling moiety being selected from the group consisting of a detectable label, a drug, or a combination thereof.

[0145] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate.

[0146] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0147] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0148] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0149] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0150] In another preferred embodiment, the drug is a small molecule drug, a biological factor, or a combination thereof.

[0151] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0152] In another preferred embodiment, the toxin is selected from the following group:

[0153] Auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), SN-38, exinotecan, Dxd, chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0154] In another preferred embodiment, the coupling moiety is MMAE.

[0155] In another preferred embodiment, the coupling moiety is a detectable label.

[0156] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0157] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0158] (ii) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0159] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold single-domain particles / single-domain rods, viral particles, liposomes, single-domain magnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of single-domain particles, etc.

[0160] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0161] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing MET protein.

[0162] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate having the structure shown in the following molecular formula:

[0163] in:

[0164] nAb is a Nanobody targeting MET as described in the first aspect of the invention, an antibody targeting MET as described in the second aspect of the invention, a Nanobody targeting EGFR as described in the third aspect of the invention, an antibody targeting EGFR as described in the fourth aspect of the invention, or a multispecific antibody as described in the fifth aspect of the invention;

[0165] LU is a chemical bond or linker;

[0166] D is for medicine;

[0167] p is the average number of drug conjugates in the antibody-drug conjugate, and p is a value selected from 1-10.

[0168] In another preferred embodiment, the average number of drug conjugates in the antibody-drug conjugate is 2 to 6, preferably 3 to 4, and more preferably 3.6 to 4.0.

[0169] In another preferred embodiment, the LU is a maleimidocaproyl-valine-citrulline (MC-Val-Cit) linker.

[0170] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0171] (i) the MET-targeting Nanobody as described in the first aspect of the invention, the MET-targeting antibody as described in the second aspect of the invention, the EGFR-targeting Nanobody as described in the third aspect of the invention, the EGFR-targeting antibody as described in the fourth aspect of the invention, the multispecific antibody as described in the fifth aspect of the invention, the recombinant protein as described in the sixth aspect of the invention, the recombinant immune cell as described in the eighth aspect of the invention, or the immunoconjugate as described in the ninth aspect of the invention;

[0172] (ii) a pharmaceutically acceptable carrier.

[0173] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0174] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0175] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0176] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0177] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0178] In another preferred embodiment, the pharmaceutical composition is an injection.

[0179] In the eleventh aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the following group: the Nanobody targeting MET as described in the first aspect of the present invention, the antibody targeting MET as described in the second aspect of the present invention, the Nanobody targeting EGFR as described in the third aspect of the present invention, the antibody targeting EGFR as described in the fourth aspect of the present invention, the multispecific antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the recombinant immune cell as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof, the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a medicament for preventing and / or treating a disease.

[0180] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0181] (1) detecting MET and / or EGFR protein in a sample; and / or

[0182] (2) Detect tumor cells expressing MET protein and / or EGFR protein.

[0183] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose MET and / or EGFR related diseases.

[0184] In another preferred embodiment, the disease is a MET and / or EGFR related disease.

[0185] In another preferred embodiment, the disease includes cancer.

[0186] In another preferred embodiment, the cancer includes solid tumors and blood cancers.

[0187] In another preferred embodiment, the cancer is selected from the group consisting of colon cancer, chromophobe renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, osteosarcoma, angiosarcoma, or a combination thereof.

[0188] In another preferred embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, and gastric cancer.

[0189] In the twelfth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the group consisting of:

[0190] (1) the MET-targeting Nanobody according to the first aspect of the invention, the MET-targeting antibody according to the second aspect of the invention, the EGFR-targeting Nanobody according to the third aspect of the invention, the EGFR-targeting antibody according to the fourth aspect of the invention, or the multispecific antibody according to the fifth aspect of the invention;

[0191] (2) the recombinant protein according to the sixth aspect of the present invention; or

[0192] (3) The CAR construct as described in the seventh aspect of the present invention.

[0193] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0194] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence selected from the following group: SEQ ID NO: 19, 20, 21, 22, or a combination thereof.

[0195] In the thirteenth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the twelfth aspect of the present invention.

[0196] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0197] In the fourteenth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector as described in the thirteenth aspect of the present invention or the polynucleotide as described in the twelfth aspect of the present invention is integrated into its genome.

[0198] In a fifteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0199] (a) culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;

[0200] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is a Nanobody targeting MET as described in the first aspect of the invention, an antibody targeting MET as described in the second aspect of the invention, a Nanobody targeting EGFR as described in the third aspect of the invention, an antibody targeting EGFR as described in the fourth aspect of the invention, or a multispecific antibody as described in the fifth aspect of the invention, or a recombinant protein as described in the sixth aspect of the invention.

[0201] In the sixteenth aspect of the present invention, a method for treating a disease is provided, the method comprising: administering to a subject in need thereof a Nanobody targeting MET as described in the first aspect of the present invention, an antibody targeting MET as described in the second aspect of the present invention, a Nanobody targeting EGFR as described in the third aspect of the present invention, an antibody targeting EGFR as described in the fourth aspect of the present invention, or a multispecific antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, a recombinant immune cell as described in the eighth aspect of the present invention, an immunoconjugate as described in the ninth aspect of the present invention, or a pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

[0202] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0203] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0204] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0206] Figure 1 shows the nanobody screening process.

[0207] FIG2 shows the binding of MET antibody KY301-01 to recombinant human MET antigen (ELISA method).

[0208] FIG3 shows the binding of MET antibody KY301-01 to recombinant human MET antigen (A), mouse MET antigen (B), and monkey MET antigen (C) (ELISA method).

[0209] FIG4 shows that the MET antibody KY301-01 can bind to CHO-K1-MET cells (CHO-K1 cells expressing human MET protein).

[0210] FIG5 shows that the MET antibody KY301-01 can be internalized in ASPC-1 (A), HCT116 (B), NCI-H1993 (C), and EBC-1 (D) cells.

[0211] FIG6 shows that the MET antibody KY301-01 can block the binding of HGF to the MET antigen.

[0212] Figure 7 shows that the MET antibody-MMAE conjugate product can effectively kill NCI-H1975 (A), HCT116 (B), BxpC-3 (C), MDA-MB-468 (D), NCI-H1993 (E), HT29 (F), MDA-MB-231 (G), and 786O cells (H).

[0213] FIG8 shows that the MET antibody-MMAE product can effectively inhibit the growth of HCT116 xenograft tumors.

[0214] Figure 9 shows that the MET antibody-MMAE product can effectively inhibit the growth of NCI-H1975 tumor cells in the NCG tumor-bearing mouse model.

[0215] FIG10 shows that the EGFR antibody KY303-58 can bind to the recombinant human EGFR antigen (ELISA method).

[0216] FIG11 shows the binding of EGFR antibody KY303-58 to recombinant human EGFR antigen (A), mouse EGFR antigen (B), and monkey EGFR antigen (C) (ELISA method).

[0217] Figure 12 shows that the EGFR antibody KY303-58 does not bind to CHO-K1 cells.

[0218] FIG13 shows that the EGFR antibody KY303-58 can be internalized in NCI-H1975 (A) and HCT116 (B) cells.

[0219] FIG14 shows that the EGFR antibody KY303-58 cannot block the binding of EGF to EGFR.

[0220] Figure 15 shows that the EGFR antibody-MMAE conjugate product can effectively kill HCT116 (A), NCI-H1975 (B), NCI-H1993 (C), MDA-MB-468 (D), MDA-MB-231 (E), HT29 (F), and BxpC-3 (G).

[0221] FIG16 shows that the EGFR antibody-MMAE product can effectively inhibit the growth of NCI-H1975 tumor cells in the NCG tumor-bearing mouse model.

[0222] FIG17 shows that the EGFR antibody-MMAE product can effectively inhibit the growth of HCT116 tumor cells in the NCG tumor-bearing mouse model.

[0223] FIG18 shows eight formats of MET-EGFR bispecific antibodies.

[0224] FIG19 shows that the MET-EGFR antibody can bind to the recombinant human MET antigen (ELISA method).

[0225] FIG20 shows that the MET-EGFR antibody can bind to the recombinant human EGFR antigen (ELISA method).

[0226] FIG21 shows that the MET-EGFR antibody can bind to recombinant human, mouse, and monkey MET and EGFR antigens.

[0227] FIG22 shows that the binding of MET-EGFR antibodies to cells is specific.

[0228] FIG23 shows that eight MET-EGFR antibody-MMAE products can effectively kill HCT116 cells.

[0229] FIG24 shows that at 1.25 μg / mL, BH01-BK58, DH01-DK58, and BH01-DK58 had significant killing effects on HCT116 cells.

[0230] FIG25 shows that eight MET-EGFR antibody-MMAE products can effectively kill NCI-H1975 cells.

[0231] FIG26 shows that at 1.25 μg / mL, BH01-BK58, DH01-DK58, and BH01-DK58 have significant killing effects on NCI-H1975 cells.

[0232] FIG27 shows the binding and dissociation kinetic curves of BH01-BK58, BH01-DK58 and the reference antibody AZ-RAA22 / B09 with MET and EGFR antigens.

[0233] FIG. 28 shows that BH01-BK58-MMAE and BH01-DK58-MMAE are mainly DAR4 products.

[0234] FIG29 shows that BH01-BK58-MMAE and BH01-DK58-MMAE can effectively kill HT29 (A), BxPC-3 (B), HS746T (C), and SNU-5 (D) cells.

[0235] Figure 30 shows that BH01-BK58-MMAE and BH01-DK58-MMAE can effectively inhibit the tumor growth of B-NDG mice inoculated with HCT116 cells, and the tumor inhibition effect of BH01-DK58-MMAE is better than that of BH01-BK58-MMAE.

[0236] Figure 31 shows that BH01-BK58-MMAE and BH01-DK58-MMAE can effectively inhibit the tumor growth of B-NDG mice inoculated with NCI-H1975 cells, and the tumor inhibition effect of BH01-DK58-MMAE is better than that of BH01-BK58-MMAE.

[0237] FIG32 shows that BH01-BK58-MMAE and BH01-DK58-MMAE were well tolerated at doses of 10 mg / kg and below.

[0238] Figure 33 shows that BH01-BK58-MMAE is somewhat toxic to male C57BL / 6 mice at doses of 16 mg / kg and 20 mg / kg, while BH01-DK58-MMAE is still well tolerated at high doses.

[0239] Figures 34A-34D show that the internalization ability of BH01-DK58 is stronger than that of single target antibodies and reference antibodies.

[0240] FIG35 shows the MET and EGFR protein levels in 9 tumor cells.

[0241] Figure 36 shows the changes in MET and EGFR protein levels and phosphorylation levels after NCI-H1975, EBC-1, and HCC827 were treated with KY-0301 1 mg / mL for 24 hours.

[0242] FIG37 shows that BH01-DK58-MMAE has a better tumor-killing effect than single-target ADCs (KY 301-01-MMAE and KY303-58-MMAE).

[0243] FIG38 shows that the toxicity of BH01-DK58-MMAE to normal cells is weaker than that of the MMAE conjugate of the reference antibody.

[0244] FIG39 shows that BH01-DK58-MMAE can induce G2 / M arrest in HCT116.

[0245] Figure 40 shows a schematic diagram of the structure of KY-0301 (i.e., BH01-DK58-MMAE).

[0246] FIG41 shows that KY-0301 can significantly inhibit the growth of human lung cancer tumor cell EBC-1 xenografts.

[0247] FIG42 shows that KY-0301 can significantly inhibit the growth of human colorectal cancer cell HT29 xenografts.

[0248] Figure 43 shows that KY-0301 has significant antitumor efficacy in the high tumor burden model and relapse model of NCI-H1975 xenograft tumor.

[0249] Figure 44 shows that KY-0301 can effectively inhibit tumor growth in a lung cancer PDX mouse model.

[0250] Figure 45 shows that KY-0301 can effectively inhibit tumor growth in a pancreatic cancer PDX mouse model.

[0251] Figure 46 shows that KY-0301 has a better inhibitory effect on human colorectal cancer cell HCT116 xenografts than AZD9592-MMAE.

[0252] FIG47 shows that KY-0301 has a better inhibitory effect on human lung cancer cell EBC-1 xenografts than AZD9592-Dxd. DETAILED DESCRIPTION

[0253] After extensive and in-depth research, the present inventors have developed for the first time an anti-MET / EGFR bispecific antibody and its drug conjugate. Specifically, the present invention provides anti-MET Nanobodies and anti-EGFR Nanobodies, as well as bispecific antibodies comprising both Nanobodies of the present invention. The bispecific Nanobodies of the present invention simultaneously target both MET and EGFR, have the characteristics of small molecular weight and rapid tumor tissue penetration, and have better toxin delivery capabilities than traditional antibodies. This is the basis for the completion of the present invention.

[0254] MET

[0255] MET, also known as the HGF (hepatocyte growth factor) receptor, consists of three regions: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain contains the Semaphorin (Semaphorin) domain, the PSI (Plexin Semaphorin Integrin) domain, and the IPT (Immunoglobulin Plexin Transcription) domain. The SPH domain of HGF interacts with the SEMA domain of MET, inducing MET activation. The intracellular region of MET comprises the juxtamembrane region, the tyrosine kinase catalytic domain, and the C-terminal tail. When HGF binds to MET, MET dimerizes, autophosphorylating tyrosine residues Y1234 and Y1235 in the tyrosine kinase catalytic domain and inducing phosphorylation of Y1349 and Y1356 in the C-terminal tail, recruiting effector molecules such as GRB2, SRC, PI3K, and GV1, activating downstream signaling pathways. Serine 975 and tyrosine Y1003 in the juxtamembrane region are important sites that negatively regulate MET activity. Phosphorylation of tyrosine Y1003 recruits the ubiquitin ligase c-CBL, which ubiquitinates MET, promoting MET degradation and reducing MET signaling. Downstream signaling pathways involved in MET include the RAS / MAPK proliferation and survival pathway, the PI3K / AKT / mTOR cell motility and invasion pathway, and the SRC / FAK mechanosensory pathway. When MET is amplified, mutated, or overexpressed, MET becomes overactivated, inducing carcinogenesis or promoting tumor resistance and invasion. For example, MET amplification is a marker of poor prognosis in non-small cell lung cancer, liver cancer, gastric cancer, and triple-negative breast cancer. In NSCLC cells resistant to EGFR inhibitors, MET gene amplification compromises treatment efficacy. MET exon 14 skipping mutations are present in approximately 4% of non-small cell lung cancers and are associated with poor prognosis in patients with these conditions responding to immunotherapy.

[0256] EGFR

[0257] EGFR, also known as HER1, is a member of the HER (Human Epidermal Growth Factor Receptor) protein family. Similar in structure to MET, it is divided into three regions: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain of EGFR consists of four domains (I, II, III, and IV). Domains I and III utilize leucine-rich structures to bind to ligands such as GF, TGF-α, and HB-EGF, while domains II and IV are rich in cysteine. The intracellular domain of EGFR is divided into a juxtamembrane region, a tyrosine kinase domain, and a C-terminal tail, containing five autophosphorylation sites: Y992, Y1045, Y1068, Y1148, and Y117. Upon ligand binding, EGFR dimers form and undergoes autophosphorylation, activating downstream RAS / RAF / MAPK, AKT, and JAK / STAT signaling pathways, regulating cell growth, proliferation, differentiation, and other physiological functions. Studies have shown that EGFR overexpression and mutations are associated with the development and progression of many tumors, as well as drug resistance. For example, elevated EGFR expression levels are a predictor of head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, etc. Exon 19 deletions and exon 21 single amino acid substitutions (L858R), known as “classic” EGFR mutations, account for approximately 85% of EGFR-mutated NSCLCs.

[0258] MET and EGFR are two important proto-oncogenes that are intertwined. Their expression is often higher in tumor tissue than in normal tissue, and they are co-expressed in various tumor cell types. As shown in the GEPIA database, the linear correlation coefficient between MET and EGFR expression is above 0.5 in colon cancer, chromophobe renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell carcinoma, and thyroid cancer, indicating a good correlation.

[0259] Table 1 Correlation between co-expression of MET and EGFR in some tumor cells

[0260] Nanobodies

[0261] As used herein, the terms "anti-MET Nanobody of the present invention" and "anti-MET Nanobody" are used interchangeably and refer to the Nanobody of the first aspect of the present invention that specifically recognizes and binds to MET (including human MET), and particularly preferably the Nanobody whose VHH chain amino acid sequence is as shown in SEQ ID NO: 1.

[0262] As used herein, the terms "anti-EGFR Nanobody of the present invention" and "anti-EGFR Nanobody" are used interchangeably and refer to the Nanobody of the first aspect of the invention that specifically recognizes and binds to EGFR (including human EGFR), and particularly preferably the Nanobody whose amino acid sequence of the VHH chain is as shown in SEQ ID NO: 3.

[0263] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0264] As used herein, the terms "nanobody" and "VHH" have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody. Nanobodies (VHH) are the smallest antigen-binding fragments with complete function. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only a single heavy chain variable region.

[0265] As used herein, the term "heavy chain antibody" refers to an antibody containing only a heavy chain. A portion of antibodies found in the blood of camelids are "heavy chain antibodies" that lack light chains. The heavy chain antibodies of the present invention comprise a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibodies of the present invention may be antibodies isolated from animals (such as camel-derived) that naturally lack light chains and heavy chain constant region 1 (CH1); or they may be recombinant antibodies obtained by recombining the nanobodies (VHH) of the present invention with heavy chain constant regions. The heavy chain antibodies of the present invention may comprise a constant region derived from, for example, IgG1, IgG2, IgG3 or IgG4, preferably a constant region derived from IgG1.

[0266] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0267] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises three complementarity determining regions (CDR1, CDR2, and CDR3). There are currently multiple CDR partitioning methods, including the IMGT method, the Kabat method, the Chothia method, the VBASE2 method, and the like. In one embodiment, the CDR partitioning methods mentioned in the present invention all use the IMGT method.

[0268] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0269] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0270] In the present invention, the terms "recombinant protein of the invention", "fusion protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to polypeptides that specifically bind to MET and / or EGFR proteins, such as proteins or polypeptides having Nanobody VHH chains of the invention. They may or may not contain an initial methionine.

[0271] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0272] Anti-MET nanobodies and anti-EGFR nanobodies

[0273] The present invention provides a Nanobody targeting MET, wherein the VHH chain of the Nanobody has the following complementarity determining regions CDR1, CDR2 and CDR3:

[0274] CDR1 shown in SEQ ID NO: 13,

[0275] CDR2 shown in SEQ ID NO: 14, and

[0276] CDR3 shown in SEQ ID NO:15.

[0277] In one embodiment, the VHH chain of the MET-targeting Nanobody has the amino acid sequence shown in SEQ ID NO: 1.

[0278] The present invention also provides a Nanobody targeting EGFR, wherein the VHH chain of the Nanobody has the following complementary determining regions CDR1, CDR2 and CDR3:

[0279] CDR1 shown in SEQ ID NO: 16,

[0280] CDR2 shown in SEQ ID NO: 17, and

[0281] CDR3 shown in SEQ ID NO:18.

[0282] In one embodiment, the VHH chain of the EGFR-targeting Nanobody has the amino acid sequence shown in SEQ ID NO:3.

[0283] Preferably, any one of the above amino acid sequences further comprises a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is capable of retaining binding affinity to MET and / or EGFR.

[0284] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0285] Wherein, the animal is preferably a mammal, such as a mouse or a camel.

[0286] In a preferred embodiment, the present invention discloses a variety of camel-derived and humanized nanobodies with high specificity and high affinity targeting MET and / or EGFR, which only include heavy chains, wherein the heavy chains contain the amino acid sequence of the heavy chain variable region (VHH) and optional constant regions CH2 and CH3.

[0287] Bispecific antibodies

[0288] The present invention also includes recombinant proteins (or fusion proteins) containing the MET and / or EGFR nanobodies of the present invention. A preferred fusion protein is a multispecific antibody, such as a bispecific antibody.

[0289] The bispecific antibodies of the present invention specifically bind to MET and EGFR antigens. In a preferred embodiment, the affinity of the bispecific antibodies of the present invention for MET (e.g., human MET) is significantly higher than the affinity for EGFR (e.g., human EGFR). For example, when the affinity is expressed as the equilibrium dissociation constant (KD value) for the antigen, the ratio of the KD value of the bispecific antibody of the present invention for EGFR to the KD value for MET (KD EGFR / KD MET )≥50, preferably ≥100, more preferably ≥150.

[0290] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0291] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, and preferably a humanized antibody.

[0292] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0293] The antibodies of the present invention may be homodimers or heterodimers. As used herein, "homodimer" refers to an antibody having two antigen-binding chains with the same amino acid sequence. As used herein, "heterodimer" refers to an antibody having two antigen-binding chains with different amino acid sequences. When the antibody of the present invention is a heterodimer, it may contain one or more specific binding sites on each chain. For example, one chain may contain a binding domain targeting Met and EGFR (such as a VHH), and the other chain may contain only a binding domain targeting Met (such as a VHH).

[0294] The bispecific antibodies of the present invention can be produced by a "knob-into-hole" strategy. In short, selected amino acids that form the constant region junction in IgG can be mutated at positions that affect the interaction of the constant regions to promote the formation of heterodimers. Amino acids with small side chains (holes) are introduced into the constant region of one chain, and amino acids with large side chains (knobs) are introduced into the constant region of the other chain. Due to the preferential interaction of the heavy chain with the "hole" and the antigen-binding chain with the "knob", heterodimers are formed between the two chains.

[0295] In each antigen-binding chain of the bispecific antibody of the present invention, the VHH portion can be connected to the Fc segment of IgG through the hinge region. In one embodiment, the EGFR-targeted VHH domain is connected to the Fc segment using a short-arm hinge. The short-arm hinge can be truncated by 1-7 amino acids at the N-terminus compared to the wild-type IgG1 hinge EPKSSDKTHTCPPCP (SEQ ID NO: 23). In one embodiment, the short-arm hinge is truncated by 4 amino acids, and the truncated hinge is mutated to ADKTHTCPPCP (SEQ ID NO: 24). The short-arm hinge design helps to reduce the antibody swing range at the EGFR end, thereby reducing the binding priority of the EGFR antibody to the antigen.

[0296] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0297] The antibodies of the present invention refer to polypeptides that have MET and / or EGFR protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties generally does not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0298] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0299] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0300] Table A

[0301] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0302] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0303] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0304] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0305] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0306] Antibody preparation

[0307] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as using PCR amplification or genomic library screening. In addition, the coding sequence of the nanobody and the constant region can be fused together to form a heavy chain antibody.

[0308] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0309] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0310] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0311] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0312] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0313] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0314] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0315] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0316] Immunoconjugates

[0317] The present invention also provides immunoconjugates (ADCs) based on the antibodies of the present invention, such as nanobody-drug conjugates (NDCs).

[0318] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0319] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0320] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0321] Nanobodies can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0322] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0323] In one embodiment, the ADCs of the invention utilize a maleimidocaproyl-valine-citrulline (MC-Val-Cit) linker.

[0324] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0325] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0326] In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0327] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0328] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0329] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0330] The ADC prepared from the bispecific antibodies provided by the present invention has a uniform drug-to-antibody ratio (DAR). In some embodiments, the DAR is in the range of 2 to 4, for example, 2 to 2.5, 2.5 to 3, 3 to 3.2, 3.2 to 3.4, 3.4 to 3.6, 3.6 to 3.8, or 3.8 to 4.

[0331] application

[0332] The present invention also provides uses of the antibodies of the present invention, such as for the preparation of diagnostic agents or medicaments for the prevention and / or treatment of MET and / or EGFR-related diseases. Such MET and / or EGFR-related diseases include tumor development, growth and / or metastasis, tumor resistance-related diseases, inflammation, and metabolic diseases.

[0333] The uses of the antibodies, ADCs, or CAR-Ts of the present invention include (but are not limited to): diagnosis, prevention, and / or treatment of tumor occurrence, growth, and / or metastasis, especially tumors with high expression of MET and / or EGFR. Such tumors include (but are not limited to): colon cancer, chromophobe renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, or a combination thereof.

[0334] Pharmaceutical composition

[0335] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0336] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0337] The pharmaceutical composition of the present invention can be used directly to bind to MET and / or EGFR protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0338] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0339] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0340] For ADC, since the nano antibody-drug conjugates provided by the present invention can target specific cell populations and bind to specific cell surface proteins (antigens), the drugs are released into the cells in an active form through endocytosis of the conjugate or drug penetration. Therefore, the nano antibody-drug conjugates of the present invention can be used to treat target diseases. The above-mentioned antibody-drug conjugates can be administered to subjects (e.g., humans) in a therapeutically effective amount through a suitable route. Subjects in need of treatment can be patients who are at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified by routine physical examinations.

[0341] When treating with the Nanobody-drug conjugates of the invention, delivery can be performed by conventional methods in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.

[0342] The main advantages of the present invention include:

[0343] 1) The nanobody of the present invention has the characteristics of small molecular weight and rapid tumor tissue penetration. Compared with traditional antibodies, the nanobody ADC constructed by it has better toxin delivery ability

[0344] 2) The MET-EGFR dual-target ADC of the present invention uses a nanobody with only two pairs of disulfide bonds in its hinge region. It can be conjugated to up to four toxins through random cysteine ​​conjugation, with uniform DAR values. The purity of the unpurified DAR4 ADC after conjugation can reach over 90%, facilitating large-scale production.

[0345] 3) The EGFR antibody of the present invention has been optimized to have moderate affinity. The affinity of the bispecific antibody of the present invention for MET is significantly higher than its affinity for EGFR. Furthermore, the MET-EGFR dual-target ADC incorporates a short-arm hinge design at the EGFR end, which, to a certain extent, reduces the binding priority between the EGFR antibody and the antigen, thereby improving the safety of the MET-EGFR dual-target ADC. In acute toxicity studies in mice, the short-arm hinge design at the EGFR end effectively reduces toxic side effects in mice.

[0346] 4) Tumor cell resistance to EGFR inhibitors is often related to the amplification of the MET gene. Simultaneous inhibition of MET and EGFR can enhance the tumor killing effect and reduce tumor resistance.

[0347] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0348] The cell lines used in the examples include:

[0349] tumor cells

[0350] Lung cancer: NCI-H1975, NCI-H1993, EBC-1, HCC827

[0351] Colorectal cancer: HCT116, HT29

[0352] Pancreatic cancer: BxPC-3, ASPC-1

[0353] Breast cancer: MDA-MB-231, MDA-MB-468

[0354] Kidney cancer: 786-O

[0355] Gastric cancer: HS746T, SNU-5

[0356] Non-tumor cells

[0357] Hamster ovary cells: CHO-K1, CHO-K1-MET (CHO-K1 cells expressing human MET protein), CHO-K1-EGFR (CHO-K1 cells expressing human EGFR protein)

[0358] Human normal tissue cells: HFL-1 (human lung fibroblasts), MIHA cells (human normal hepatocytes), MRC-5 (human embryonic lung fibroblasts), BEAS-2B (human normal bronchial epithelial cells)

[0359] Example 1. Alpaca immunization and MET nanobody screening

[0360] Alpacas were immunized with recombinant human MET antigen. After four immunizations, when the serum titer reached the threshold for library construction, mRNA was extracted from PBMCs and reverse-transcribed to obtain cDNA, which was then screened for antibodies using phage display technology. The nanobody screening process is shown in Figure 1.

[0361] Example 2. Expression and purification of MET nanobody

[0362] Through phage screening technology, the MET nanobody sequence KY301-VHH01 was obtained, and its amino acid sequence and nucleotide sequence are shown in Table 2. KY301-VHH01 was then fused with the IgG1 Fc sequence for expression, and the fusion-expressed MET nanobody was named KY301-01.

[0363] The KY301-01 CDR sequence (IMGT) is shown below:

[0364] CDR1:GRTDSTYA(SEQ ID NO:13);

[0365] CDR2:ISWSGGST(SEQ ID NO:14);

[0366] CDR3: AADHRTGSTRFRTAQYDYDY (SEQ ID NO: 15).

[0367] Table 2 Amino acid and nucleic acid sequences of the variable regions of MET antibodies

[0368] Example 3. Evaluation of Binding Activity of MET Antibodies to Human MET Antigen

[0369] method:

[0370] 1. Coat with recombinant human MET antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0371] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0372] 3. Add MET antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0373] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0374] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0375] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0376] The experimental results are shown in Table 3 and Figure 2. The results show that the MET antibody KY301-01 can bind to the MET antigen, with antigen-level binding activity comparable to that of MET-BMK2 and MET-BMK4. MET-BMK2 is derived from SEQ ID NO:82 in patent US11142578B2, and MET-BMK4 is derived from the MET monoclonal antibody portion of Amivantamab.

[0377] Table 3 EC of MET antibody binding to recombinant human MET antigen 50 value

[0378] Example 4. Evaluation of species cross-binding activity of MET antibodies

[0379] method:

[0380] 1. Coat the recombinant human, mouse, and monkey MET antigens at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0381] Block with 300 μL / well of 2.3% BSA solution for 2 h, and wash twice with 200 μL PBST (0.01% Tween-20);

[0382] 3. Add MET antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0383] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0384] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0385] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0386] As shown in Figure 3 and Table 4, the experimental results show that the MET antibody KY301-01 can bind to human, mouse, and monkey MET antigens, showing species cross-reactivity. MET-BMK2 and MET-BMK4 have no binding activity to the mouse MET antigen.

[0387] Table 4 EC of MET antibodies binding to mouse and monkey MET antigens 50 value

[0388] Example 5. Evaluation of MET Antibody Binding Activity at the Cellular Level

[0389] method:

[0390] 1. CHO-K1-MET cells (CHO-K1 cells expressing human MET protein) were plated into 96-well clear V-bottom plates at 1E5 cells / well and 100 μL of cell suspension per well.

[0391] 2. Add 100 μL of diluted test antibody solution to each well, incubate at 4°C for 1 hour, and wash the cells twice with 200 μL of 1% FBS-containing PBS;

[0392] 3. Add 100 μL of 1:100 diluted anti-Fc-PE antibody to each well and incubate at 4°C for 0.5 h.

[0393] 4. Wash the cells twice with 100 μL of PBS containing 1% FBS, resuspend the cells in 100 μL, detect the fluorescence signal in the PE fluorescence channel, and observe the median fluorescence intensity.

[0394] As shown in Figure 4, the experimental results showed that the MET antibody had binding activity with CHO-K1-MET cells, and the binding activity was stronger than that of MET-BMK2.

[0395] Example 6. Evaluation of the endocytic activity of MET antibodies in tumor cells

[0396] method:

[0397] 1. ASPC-1, HCT116, NCI-H1993, and EBC-1 cells were plated into 96-well clear V-bottom plates at 3E5 cells / well, with 100 μL of cell suspension per well.

[0398] 2. Add 100 μL of diluted test antibody solution to each well, incubate at 4°C for 1 hour, and wash the cells twice with 200 μL of culture medium;

[0399] 3. Resuspend the cells in 200 μL of culture medium and transfer 100 μL of cell suspension to each well of a new 96-well V-bottom plate. Incubate at 37°C for 2 hours. Incubate the remaining cells in the plate at 4°C for 2 hours.

[0400] 4. Centrifuge and remove the supernatant. Add 100 μL of 1:100 diluted anti-Fc-PE antibody to each well and incubate at 4°C for 0.5 h.

[0401] 5. Wash the cells twice with 100 μL of PBS containing 1% FBS, resuspend the cells in 100 μL, detect the fluorescence signal in the PE fluorescence channel, observe the median fluorescence intensity, and calculate the endocytosis rate.

[0402] As shown in Figure 5 , the experimental results showed that MET antibody was internalized in ASPC-1, HCT116, NCI-H1993, and EBC-1 cells, and the internalization rate was related to the cell type.

[0403] Example 7. Evaluation of ligand blocking activity of MET antibodies

[0404] method:

[0405] 1. Coat with recombinant human MET antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0406] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0407] 3. Add 100 μL of a mixture of MET antibody and different gradient dilutions of HA-tagged HGF ligand, incubate at room temperature for 1 h, and wash twice with 200 μL of PBST (0.01% Tween-20);

[0408] 4. Add 100 μL of anti-HA-HRP antibody diluent, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0409] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0410] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0411] As shown in FIG6 , the experimental results show that KY301-01 has the effect of blocking HGF.

[0412] Example 8. MET Antibody Conjugated to MMAE

[0413] method:

[0414] 1. Add histidine-acetate buffer (pH 5.5), EDTA-2Na solution, TCEP solution, and MET antibody in sequence to make the final concentration of MET antibody 2-3 mg / mL, the final concentration of EDTA-2Na 5 mM, and the amount of TCEP 8 times the amount of MET antibody. Mix well and shake at 37°C for 4 h.

[0415] 2. Add 9 times the amount of MMAE and DMSO solution as the antibody substance, so that the total amount of DMSO accounts for 10% of the total reaction volume, and shake the reaction at 4°C for 2 hours;

[0416] 3. Add 7 times the amount of L-cysteine ​​as the antibody substance and mix well;

[0417] 4. Centrifuge the reaction product, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge, add histidine-acetate buffer, and centrifuge again. Repeat 18 times to remove free toxins not coupled to the antibody.

[0418] 5. Remove the liquid from the ultrafiltration tube, rinse the ultrafiltration tube with histidine-acetate buffer, mix with the previously removed liquid, measure the absorbance at 280 nm, and calculate the antibody concentration.

[0419] Example 9. Cytotoxicity Evaluation of MET Antibody-MMAE Product

[0420] method:

[0421] 1. NCI-H1975 / HCT116 / BxPC-3 / MDA-MB-468 / NCI-H1993 / HT29 / MDA-MB-231 / 786O cells were plated into black-walled, clear-bottomed 96-well plates, with 100 μL of cell suspension per well.

[0422] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0423] 3. Add 100 μL of CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and measure the fluorescence intensity.

[0424] As shown in Figure 7, the experimental results show that the MET antibody-MMAE conjugate has different sensitivities to different tumor cells and can effectively inhibit the growth of various tumor cells at high concentrations.

[0425] Table 5 IC50 values ​​(μg / mL) of MET antibody-MMAE conjugate products in different tumor cells

[0426] Example 10. MET Antibody-MMAE Product Inhibits the Growth of HCT116 Xenograft Tumors

[0427] method:

[0428] 1. HCT116 was inoculated subcutaneously in the right anterior flank of female NCG mice (5-6 weeks old, 18-21 days);

[0429] 2. Wait until the tumor grows to 100-150mm 3 When left and right, divide them into 3 groups, 5 in each group;

[0430] 3. Administer MET single-target ADC or PBS via tail vein injection at a dose of 6 mg / kg or 2 mg / kg, once a week for 2 weeks;

[0431] 4. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0432] 5. Calculation of tumor volume and tumor growth inhibition rate:

[0433] Tumor volume: (V) was calculated as (length × width 2 ) / 2

[0434] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0435] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0436] As shown in Figure 8, the experimental results showed that in the NCG mouse model inoculated with HCT116 tumor cells, tumor growth was significantly inhibited at a dose of 2 mg / kg, with a tumor inhibition rate of 79.1% on Day 16. At a higher dose (6 mg / kg), the inhibitory effect of the MET single-target ADC on tumor volume was further significantly improved, with a tumor inhibition rate of 98.6%. These results indicate that the MET single-target ADC can effectively inhibit the growth of HCT116 xenografts in a dose-dependent manner. At the same time, neither the low-dose nor the high-dose groups showed a significant effect on mouse body weight.

[0437] Example 11. MET Antibody-MMAE Product Inhibits Growth of NCI-H1975 Xenograft Tumors

[0438] method:

[0439] 1. NCI-H1975 cells were inoculated subcutaneously in the right anterior flank of female NCG mice (5-6 weeks old, 18-21 days old);

[0440] 2. Wait until the tumor grows to 100-150mm 3When left and right, divide them into 3 groups, 5 in each group;

[0441] 3. Administer MET single-target ADC or PBS via tail vein injection at a dose of 6 mg / kg or 2 mg / kg once a week for 2 weeks;

[0442] 4. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0443] 5. Calculation of tumor volume and tumor growth inhibition rate:

[0444] Tumor volume: (V) is calculated as (length × width 2 ) / 2

[0445] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0446] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0447] As shown in Figure 9, experimental results show that in the NCG mouse model inoculated with NCI-H1975 tumor cells, tumor growth was significantly inhibited at both 2 mg / kg and 6 mg / kg doses, with Day 20 tumor inhibition rates of 97.6% and 98.01%, respectively. These results demonstrate that the MET single-target ADC can effectively inhibit the growth of HNCI-H1975 xenograft tumors in a dose-dependent manner. Furthermore, neither the low-dose nor the high-dose groups showed a significant effect on mouse body weight.

[0448] Example 12. Alpaca immunization and EGFR nanobody screening, expression and purification

[0449] Alpacas were immunized with recombinant human EGFR antigen. After four immunizations, when the serum titer reached the threshold for library construction, mRNA was extracted from PBMCs and reverse-transcribed to obtain cDNA. Antibody screening was performed using phage display technology. The nanobody screening process is shown in Figure 1.

[0450] After phage screening, the EGFR nanobody sequence KY303-VHH58 was obtained, and its amino acid sequence and nucleotide sequence are shown in Table 6. KY303-VHH58 was then fused with the IgG1 Fc sequence for expression, and the fusion-expressed EGFR nanobody was named KY303-58.

[0451] The CDR sequence of KY303-58 (IMGT) is shown below:

[0452] CDR1:GSTFSSYA(SEQ ID NO:16);

[0453] CDR2: ISSGGST (SEQ ID NO: 17);

[0454] CDR3: NLHPRVN (SEQ ID NO: 18).

[0455] Table 6 Variable region amino acid sequence and nucleic acid sequence of EGFR antibodies

[0456] Example 13. Evaluation of Binding Activity of EGFR Antibodies to Human EGFR Antigen

[0457] method:

[0458] 1. Coat with recombinant human EGFR antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0459] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0460] 3. Add EGFR antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0461] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0462] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0463] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0464] As shown in Figure 10, the experimental results show that the EGFR antibody KY303-58 can bind to the EGFR antigen with lower affinity than the benchmark antibody EGFR-BMK2, indicating a weaker affinity for EGFR, suggesting a better safety profile. EGFR-BMK2 is derived from SEQ ID NO 34 and SEQ ID NO 42 in patent US10047160B2.

[0465] Table 7 EC of EGFR antibody binding to recombinant human EGFR antigen 50 value

[0466] Example 14. Evaluation of species cross-binding activity of EGFR antibodies

[0467] method:

[0468] 1. Coat the recombinant human, mouse, and monkey EGFR antigens at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0469] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0470] 3. Add EGFR antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0471] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0472] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0473] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0474] As shown in Figure 11, the experimental results show that the EGFR antibody KY303-58 can bind to human, mouse, and monkey EGFR antigens, showing species cross-reactivity. EGFR-BMK1 has no binding activity to mouse EGFR antigen. EGFR-BMK1 is derived from Cetuximab.

[0475] Table 8 EC of EGFR antibodies binding to mouse and monkey EGFR antigens 50 value

[0476] Example 15. Binding specificity of EGFR antibodies at the cellular level

[0477] method:

[0478] 1. CHO-K1 and CHO-K1-EGFR cells (CHO-K1 cells expressing human EGFR protein) were plated into 96-well clear V-bottom plates at 1E5 cells / well with 100 μL of cell suspension per well.

[0479] 2. Add 100 μL of diluted test antibody solution to each well, incubate at 4°C for 1 hour, and wash the cells twice with 200 μL of 1% FBS-containing PBS;

[0480] 3. Add 100 μL of 1:100 diluted anti-Fc-PE antibody to each well and incubate at 4°C for 0.5 h.

[0481] 4. Wash the cells twice with 100 μL of PBS containing 1% FBS, resuspend the cells in 100 μL, detect the fluorescence signal in the PE fluorescence channel, and observe the median fluorescence intensity.

[0482] As shown in FIG12 , the experimental results showed that the EGFR antibody did not bind to CHO-K1 cells, indicating that the binding of the EGFR antibody at the cellular level was specific.

[0483] Example 16. Evaluation of endocytic activity of EGFR antibodies in tumor cells

[0484] method:

[0485] 1. HCT116 and NCI-H1975 cells were plated into 96-well clear V-bottom plates at a density of 3E5 cells per well and 100 μL of cell suspension per well.

[0486] 2. Add 100 μL of diluted test antibody solution to each well, incubate at 4°C for 1 hour, and wash the cells twice with 200 μL of culture medium;

[0487] 3. Resuspend the cells in 200 μL of culture medium and transfer 100 μL of cell suspension to each well of a new 96-well V-bottom plate. Incubate at 37°C for 2 hours. Incubate the remaining cells in the plate at 4°C for 2 hours.

[0488] 4. Centrifuge and remove the supernatant. Add 100 μL of 1:100 diluted anti-Fc-PE antibody to each well and incubate at 4°C for 0.5 h.

[0489] 5. Wash the cells twice with 100 μL of PBS containing 1% FBS, resuspend the cells in 100 μL, detect the fluorescence signal in the PE fluorescence channel, observe the median fluorescence intensity, and calculate the endocytosis rate.

[0490] As shown in FIG13 , the experimental results showed that EGFR antibodies were internalized in both HCT116 and NCI-H1975 cells, and the internalization rate was related to the cell type.

[0491] Example 17. Evaluation of ligand blocking activity of EGFR antibodies

[0492] method:

[0493] 1. Coat with recombinant human EGFR antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0494] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0495] 3. Add 100 μL of a mixture of EGFR antibody and different gradient dilutions of HA-tagged EGF ligand, incubate at room temperature for 1 hour, and wash twice with 200 μL of PBST (0.01% Tween-20);

[0496] 4. Add 100 μL of anti-HA-HRP antibody diluent, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0497] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0498] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0499] As shown in FIG14 , the experimental results showed that KY303-58 did not block the binding of EGF to EGFR.

[0500] Example 18. EGFR Antibody Conjugated to MMAE

[0501] method:

[0502] 1. Add histidine-acetate buffer (pH 5.5), EDTA-2Na solution, TCEP solution, and EGFR antibody in sequence to make the final concentration of EGFR antibody 2-3 mg / mL, the final concentration of EDTA-2Na 5 mM, and the amount of TCEP 8 times the amount of EGFR antibody. Mix well and shake at 37°C for 4 h.

[0503] 2. Add 9 times the amount of MMAE and DMSO solution as the antibody substance, so that the total amount of DMSO accounts for 10% of the total reaction volume, and shake the reaction at 4°C for 2 hours;

[0504] 3. Add 7 times the amount of L-cysteine ​​as the antibody substance and mix well;

[0505] 4. Centrifuge the reaction product, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge, add histidine-acetate buffer, and centrifuge again. Repeat 18 times to remove free toxins not coupled to the antibody.

[0506] 5. Remove the liquid from the ultrafiltration tube, rinse the ultrafiltration tube with histidine-acetate buffer, mix with the previously removed liquid, measure the absorbance at 280 nm, and calculate the antibody concentration.

[0507] Example 19. Cytotoxicity Evaluation of EGFR Antibody-MMAE Product

[0508] method:

[0509] 1. NCI-H1975 / HCT116 / BxPC-3 / MDA-MB-468 / NCI-H1993 / HT29 / MDA-MB-231 cells were plated into black-walled, clear-bottomed 96-well plates, with 100 μL of cell suspension per well.

[0510] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0511] 3. Add 100 μL of CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and measure the fluorescence intensity.

[0512] As shown in FIG15 and Table 9, EGFR antibody-MMAE can effectively inhibit the growth of various tumor cells.

[0513] Table 9 IC50 values ​​(μg / mL) of EGFR antibody-MMAE conjugate products in different tumor cells

[0514] Example 20. EGFR Antibody-MMAE Product Effectively Inhibits the Growth of NCI-H1975 Xenograft Tumors

[0515] method:

[0516] 1. NCI-H1975 cells were subcutaneously inoculated in the right anterior flank of 15 female NCG mice;

[0517] 2. Wait until the tumor grows to 100-150mm 3 Around 2 d, the mice were randomly divided into 3 groups, 5 mice in each group;

[0518] 3. Inject PBS and EGFR single-target ADC into the tail vein at a dose of 1.5 mg / kg and 3 mg / kg, once a week for 2 weeks;

[0519] 4. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time.

[0520] As shown in Figure 16, the experimental results showed that at doses of 1.5 mg / kg and 3 mg / kg, tumor growth inhibition rates on Day 14 were 89.5% and 93.5%, respectively. These results demonstrate that the EGFR single-target ADC significantly inhibited the growth of NCI-H1975 xenograft tumors. Furthermore, neither the low-dose nor the high-dose groups showed a significant effect on mouse body weight.

[0521] Example 21. Method for effectively inhibiting the growth of HCT116 xenograft tumors using EGFR antibody-MMAE products:

[0522] 1. HCT116 cells were subcutaneously inoculated in the right anterior flank of 15 female NCG mice;

[0523] 2. Wait until the tumor grows to 100-150mm 3 Around 2 d, the mice were randomly divided into 3 groups, 5 mice in each group;

[0524] 3. Inject PBS and EGFR single-target ADC into the tail vein. The dosage of EGFR single-target ADC was 0.5 mg / kg, 1.5 mg / kg, and 4.5 mg / kg, once a week for 2 weeks.

[0525] 4. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time.

[0526] As shown in Figure 17, the experimental results showed that at a dose of 4.5 mg / kg, the tumor growth inhibition rate on Day 18 was 60.2%. These results indicate that the EGFR single-target ADC can inhibit the growth of HCT116 xenograft tumors. Furthermore, the low-, medium-, and high-dose groups did not show a significant effect on mouse body weight.

[0527] Example 22. Design of MET-EGFR dual antibody

[0528] Eight types of MET and EGFR combination bispecific antibodies were designed: BH01-BK58, DH01-DK58, BH01-DK58, BH01-BK0158, BH01-BK5801, BH01-BK01-C58, 01-58-IgG1, and 01-IgG1-58. 01-58-IgG1 and 01-IgG1-58 utilize wild-type IgG1 Fc; the Fc regions of BH, BK, DH, and DK are mutated to form either hole or knob chains, creating a "knob-into-hole" link between the two Fc chains. BH01-BK58, DH01-DK58 and BH01-DK58 are "1+1" "Y" type, approximately symmetrical structures, where "DH" and "DK" are hinge truncated forms of "BH" and "BK"; BH01-BK0158 and BH01-BK5801 are "1+2" ​​"Y" type structures, where both the BK0158 arm and the BK5801 arm have CDR regions that bind to MET and EGFR, differing only in their front and back positions; in BH01-BK01-C58, the EGFR-binding arm is located at the other end of the Fc of BH01-BK58; 01-58-IgG1 is a "2+2" "Y" type, approximately symmetrical structure, where each Fab has CDR regions that bind to MET and EGFR; 01-IgG1-58 has four Fabs, located on both sides of the Fc, and the CDR region sequences of the Fabs on the same side are identical. The eight formats of MET-EGFR dual antibodies are shown in FIG18 , and the amino acid sequences of each arm are shown in Table 10 .

[0529] Table 10 Amino acid sequence of MET-EGFR dual antibody

[0530] Example 23. Transient expression of MET-EGFR dual antibody

[0531] method:

[0532] 1. Transient transfection of 293T cells with BH01-BK58 plasmids (co-transfection of BH01 and BK58 plasmids), DH01-DK58 (co-transfection of DH01 and DK58 plasmids), BH01-DK58 (co-transfection of BH01 and DK58 plasmids), BH01-BK0158 (co-transfection of BH01 and BK0158 plasmids), BH01-BK5801 (co-transfection of BH01 and BK5801 plasmids), BH01-BK01-C58 (co-transfection of BH01 and BK01C58 plasmids), 01-58-IgG1 (single plasmid transfection of 01-58-IgG1), and 01-IgG1-58 (single plasmid transfection of 01-IgG1-58).

[0533] 2. Lyse the cells, perform protein A affinity purification, collect the eluate and concentrate.

[0534] Table 11 Expression levels and purity of 8 MET-EGFR dual antibodies

[0535] Example 24. Binding activity of MET-EGFR dual antibody and recombinant human MET antigen

[0536] method:

[0537] 1. Coat with recombinant human MET antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0538] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0539] 3. Add MET-EGFR dual antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0540] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0541] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0542] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0543] As shown in Figure 19 and Table 12, the experimental results showed that the MET-EGFR dual antibody can bind to the MET antigen.

[0544] Table 12 EC of MET-EGFR antibody binding to recombinant human MET antigen 50 value

[0545] Example 25. Binding activity of MET-EGFR dual antibody and recombinant human EGFR antigen

[0546] method:

[0547] 1. Coat with recombinant human EGFR antigen at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0548] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0549] 3. Add MET-EGFR dual antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0550] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0551] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0552] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0553] As shown in Figure 20 and Table 13, the experimental results showed that the MET-EGFR bispecific antibodies could bind to the EGFR antigen, among which BH01-BK58, DH01-DK58, BH01-DK58, and 01-58-IgG1 had stronger binding ability to hEGFR.

[0554] Table 13 EC of MET-EGFR antibody binding to recombinant human EGFR antigen 50 value

[0555] Example 26. Method for determining species cross-reactivity of candidate MET-EGFR bispecific antibodies against MET and EGFR antigens:

[0556] 1. Coat the recombinant human, mouse, or monkey MET or EGFR antigens at 4°C overnight, then wash twice with 200 μL PBST (0.01% Tween-20);

[0557] 2. Block with 300 μL / well of 3% BSA solution for 2 h, then wash twice with 200 μL PBST (0.01% Tween-20);

[0558] 3. Add MET-EGFR dual antibody, incubate at room temperature for 1 hour, and wash twice with 200 μL PBST (0.01% Tween-20);

[0559] 4. Add 100 μL of anti-Fc-HRP antibody dilution solution, incubate at 37°C for 0.5 h, and wash four times with 200 μL of PBST (0.01% Tween-20);

[0560] 5. Add 100 μL TMB colorimetric solution and allow to develop for 5 minutes;

[0561] 6. Add 50 μL of stop solution to terminate the reaction and read the absorbance at 450 nm on a microplate reader.

[0562] As shown in Figure 21 and Tables 14-15, the experimental results show that the MET-EGFR bispecific antibody can bind to the MET antigen of mice and monkeys, and has species cross-reactivity.

[0563] Table 14 EC of MET-EGFR antibodies binding to recombinant mouse and monkey MET antigens 50 Value (μg / mL)

[0564] Table 15 EC of MET-EGFR antibody binding to recombinant mouse and monkey EGFR antigens 50 Value (μg / mL)

[0565] Example 27. Cell Binding Specificity of Candidate MET-EGFR Dual Antibodies

[0566] method:

[0567] 1. CHO-K1, CHO-K1-EGFR cells (CHO-K1 cells expressing human EGFR protein), and CHO-K1-MET cells (CHO-K1 cells expressing human MET protein) were plated into 96-well clear V-bottom plates at 1E5 cells / well, with 100 μL of cell suspension per well.

[0568] 2. Add 100 μL of diluted test antibody solution to each well, incubate at 4°C for 1 hour, and wash the cells twice with 200 μL of 1% FBS-containing PBS;

[0569] 3. Add 100 μL of 1:100 diluted anti-Fc-PE antibody to each well and incubate at 4°C for 0.5 h.

[0570] 4. Wash the cells twice with 100 μL of PBS containing 1% FBS, resuspend the cells in 100 μL, detect the fluorescence signal in the PE fluorescence channel, and observe the median fluorescence intensity.

[0571] As shown in Figure 22, the experimental results showed that the MET-EGFR dual antibody did not bind to CHO-K1 antigen-negative cells, but bound to CHO-K1 antigen-positive cells (CHO-K1-EGFR, CHO-K1-MET), indicating that its binding at the cellular level was specific.

[0572] Example 28. MET-EGFR dual antibody coupled with MMAE

[0573] method:

[0574] 1. Add histidine-acetate buffer (pH 5.5), EDTA-2Na solution, TCEP solution, and EGFR antibody in sequence to make the final concentration of MET-EGFR antibody 2-3 mg / mL, the final concentration of EDTA-2Na 5 mM, and the amount of TCEP 8 times the amount of MET-EGFR antibody. Mix well and shake at 37°C for 4 h.

[0575] 2. Add 9 times the amount of MMAE and DMSO solution as the antibody substance, so that the total amount of DMSO accounts for 10% of the total reaction volume, and shake the reaction at 4°C for 2 hours;

[0576] 3. Add 7 times the amount of L-cysteine ​​as the antibody substance and mix well;

[0577] 4. Centrifuge the reaction product, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge, add histidine-acetate buffer, and centrifuge again. Repeat 18 times to remove free toxins not coupled to the antibody.

[0578] 5. Remove the liquid from the ultrafiltration tube, rinse the ultrafiltration tube with histidine-acetate buffer, mix with the previously removed liquid, measure the absorbance at 280 nm, and calculate the antibody concentration.

[0579] Example 29. MET-EGFR Antibody-MMAE Product More Effectively Inhibits HCT116 Tumor Cell Growth Than Single-Target Antibody-MMAE

[0580] method:

[0581] 1. HCT116 cells were plated into a black-walled, clear-bottomed 96-well plate, with 100 μL of cell suspension per well.

[0582] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0583] 3. Add 100 μL of CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and measure the fluorescence intensity.

[0584] As shown in Figure 23, the experimental results show that MET-EGFR antibody-MMAE can effectively inhibit the growth of HCT116 tumor cells. Among them, BH01-BK58-MMAE, DH01-DK58-MMAE, and BH01-DK58-MMAE (KY-0301) have better tumor killing effects and can effectively inhibit tumor growth at low concentrations. As shown in Figure 24 and Table 16, at the same dose, the bispecific MET-EGFR antibody conjugate BH01-DK58-MMAE (KY-0301) of the present invention has significantly better cell killing performance against HCT116 target cells than MET single-target ADC (KY301-01-MMAE) and EGFR single-target ADC (KY303-58-MMAE), showing a synergistic effect.

[0585] Table 16. Cell viability of HCT116 at 1.25 μg / mL ADC concentration

[0586] Example 30. MET-EGFR Antibody-MMAE Product More Effectively Inhibits the Growth of NCI-H1975 Tumor Cells than Single-Target Antibody-MMAE

[0587] method:

[0588] 1. NCI-H1975 cells were plated into a black-walled, clear-bottomed 96-well plate, with 100 μL of cell suspension per well.

[0589] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0590] 3. Add 100 μL of CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and measure the fluorescence intensity.

[0591] As shown in Figure 25, the experimental results show that MET-EGFR antibody-MMAE can effectively inhibit the growth of HCT116 tumor cells. Among them, BH01-BK58-MMAE, DH01-DK58-MMAE, BH01-DK58-MMAE, BK01-BH0158-MMAE, and BK01-BH5801-MMAE have better tumor killing effects. As shown in Figure 26 and Table 17, at the same dose, the bispecific MET-EGFR antibody conjugate BH01-DK58-MMAE (KY-0301) of the present invention has significantly better cell killing performance against NCI-H1975 target cells than MET single-target ADC (KY301-01-MMAE) and EGFR single-target ADC (KY303-58-MMAE), showing a synergistic effect.

[0592] Table 17. Cell viability of NCI-H1975 at 1.25 μg / mL ADC concentration

[0593] Example 31. Binding affinity and kinetic constants of BH01-BK58 and BH01-DK58

[0594] The equilibrium dissociation constants (KD values) of MET and EGFR binding to MET-EGFR antibodies were determined using a real-time surface plasmon resonance biosensor assay on a Biacore 2000 instrument.

[0595] method:

[0596] 1. Antibody capture: Using 1× PBST (1× PBS solution containing 0.05% Tween 20, pH 7.4) buffer as the running buffer, dilute each antibody to 1 μg / mL in PBS buffer, set the flow rate to 10 μL / min, and capture the antibody with the Protein A chip for 60 seconds;

[0597] 2. Binding monitoring: Human recombinant MET antigen was diluted with 1× PBST to different concentrations (0 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, and 2000 nM) and injected onto the antibody capture surface at a flow rate of 30 μL / min. The binding of the antigen to the antibody was monitored for 120 s.

[0598] 3. Dissociation monitoring: Monitor MET antigen dissociation in 1× PBST running buffer for 360 seconds;

[0599] 4. Regeneration: Use Gly-HCl buffer (pH 1.5) for regeneration at a flow rate of 30 μL / min for 30 seconds, and stabilize the chip with 1× PBST for 60 seconds.

[0600] 5. Antibody capture: Using 1× PBST (1× PBS solution containing 0.05% Tween 20, pH 7.4) buffer as the running buffer, dilute each antibody to 1 μg / mL in PBS buffer, set the flow rate to 10 μL / min, and capture the antibody with the Protein A chip for 60 seconds;

[0601] 6. Binding monitoring: Human recombinant EGFR antigen was diluted in 1× PBST to different concentrations (0 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, and 2000 nM) and injected onto the antibody capture surface at a flow rate of 30 μL / min. The binding of the antigen to the antibody was monitored for 120 s.

[0602] 7. Dissociation monitoring: Monitor MET antigen dissociation in 1× PBST running buffer for 360 s;

[0603] 8. Regeneration: Use Gly-HCl buffer (pH 1.5) for regeneration, set the flow rate to 30 μL / min, and regenerate for 30 s.

[0604] The binding and dissociation kinetics of BH01-BK58, BH01-DK58, and the target antibody AZ-RAA22 / B09 are shown in Figure 27. The affinity constants are summarized in Table 18. The K values ​​of BH01-DK58 and BH01-BK58 for hMET are similar, twice the affinity of the MET antibody KY301-01, indicating that the affinity of BH01-DK58 and BH01-BK58 for hMET is weaker than that of KY301-01. A comparison of their affinity for hEGFR reveals that the affinity of BH01-DK58 and BH01-BK58 for hMET is ranked from strongest to weakest: KY303-58, BH01-BK58, and BH01-DK58. BH01-DK58 and BH01-BK58 have the same VHH sequence. The difference in their affinity to hEGFR stems from the "short-arm" hinge design of DK58, which reduces the cytotoxicity to normal tissue cells caused by targeting EGFR.

[0605] Table 18 Binding affinity of MET-EGFR bispecific antibodies to recombinant human MET or EGFR antigens

[0606] As shown in Tables 19 and 20, the affinity of BH01-DK58 for hMET and hEGFR differs by 161-fold, far exceeding the affinity difference of the benchmark antibody AZ-RAA22 / B09 for hMET and hEGFR (a 24.7-fold difference in affinity for hMET and hEGFR). This differential affinity for hMET and hEGFR is designed to reduce the binding priority of the MET-EGFR antibody to EGFR, further reducing the toxic side effects caused by targeting EGFR and improving safety.

[0607] Table 19 Binding affinity of BH01-DK58 to recombinant human, mouse, monkey MET or EGFR

[0608] Table 20 Binding affinity of the benchmark MET-EGFR dual antibody (AZ-RAA22 / B09) to recombinant human, mouse, and monkey MET or EGFR

[0609] Example 32. BH01-BK58 and BH01-DK58 dual antibodies coupled with MMAE

[0610] method:

[0611] 1. Add histidine-acetate buffer (pH 5.5), EDTA-2Na solution, TCEP solution, and EGFR antibody in sequence to make the final concentration of MET-EGFR antibody 2-3 mg / mL, the final concentration of EDTA-2Na 5 mM, and the amount of TCEP 8 times the amount of MET-EGFR antibody. Mix well and shake at 37°C for 4 h.

[0612] 2. Add 9 times the amount of MMAE and DMSO solution as the antibody substance, so that the total amount of DMSO accounts for 10% of the total reaction volume, and shake the reaction at 4°C for 2 hours;

[0613] 3. Add 7 times the amount of L-cysteine ​​as the antibody substance and mix well;

[0614] 4. Centrifuge the reaction product, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge, add histidine-acetate buffer, and centrifuge again. Repeat 18 times to remove free toxins not coupled to the antibody.

[0615] 5. Remove the liquid from the ultrafiltration tube, rinse the ultrafiltration tube with histidine-acetate buffer, mix with the previously removed liquid, measure the absorbance at 280 nm, and calculate the antibody concentration.

[0616] Example 33. DAR value and free toxin detection of BH01-BK58 and BH01-DK58 dual antibody conjugated MMAE products

[0617] method:

[0618] DAR value detection:

[0619] 50 μg of the BH01-BK58 and BH01-DK58 dual antibody conjugated MMAE products were injected into an HPLC column and eluted with different mobile phases. The chromatograms were recorded simultaneously ( Figure 28 ). The DAR value was calculated according to DAR = Σ (relative peak area × number of loaded drugs) / 100.

[0620] Free toxin detection:

[0621] 80 μg of the conjugated sample was mixed with 3 μL of DMSO and then mixed with 60 μL of anhydrous methanol-acetonitrile mixture containing sodium chloride. 20 μL was injected into a high-performance liquid chromatography (HPLC) machine for elution and the chromatogram was recorded. The free toxin content in the analyte was calculated by fitting a regression equation based on the peak area values ​​corresponding to different concentrations of the toxin standard: Free Drug (mol / mol%) = Residual Small Molecule Molar Concentration / Antibody Molar Concentration × 100.

[0622] Table 21 DAR values ​​and free toxin content of MET-EGFR antibody-MMAE products

[0623] Table 22 DAR value product content of MET-EGFR antibody-MMAE product

[0624] Example 34. BH01-BK58-MMAE and BH01-DK58-MMAE's ability to kill tumor cells

[0625] method:

[0626] 1. BxPC-3 / HT29 / HS746T / SNU-5 cells were plated into a black-walled, clear-bottomed 96-well plate, with 100 μL of cell suspension per well.

[0627] 2. Add 10 μL of serially diluted test antibody to each well and incubate for 6 days;

[0628] 3. Add 100 μL of CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and measure the fluorescence intensity.

[0629] As shown in Figure 29, both BH01-BK58-MMAE and BH01-DK58-MMAE can effectively kill BxPC-3 / HT29 / HS746T / SNU-5 tumor cells, and the killing effects are similar.

[0630] Example 35. The anti-tumor efficacy of BH01-DK58-MMAE and BH01-BK58-MMAE on HCT116 xenograft tumors is better than that of BH01-DK58-MMAE.

[0631] Experimental mice: Female B-NDG mice, 36 at the time of the experiment, 7 weeks old;

[0632] Experimental cells: Human colon cancer tumor cells HCT116 in the logarithmic growth phase were collected, the culture medium was removed and the cells were washed twice with PBS before inoculation;

[0633] Inoculum size: 5×10 6 / 100μL / mouse;

[0634] Inoculation site: right back area;

[0635] Grouped drug administration: When the average tumor volume grows to 100-150mm 3 Around 24 hours, the tumor-bearing mice were randomly divided into groups and the drug was administered on the day of grouping, which was defined as Day 0. The drug was administered intravenously once a week for a total of 2 times. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. The tumor volume (mm 3) is calculated as (length × width 2 ) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0636] Dosage: 0.5 mg / kg, 1 mg / kg, 3 mg / kg;

[0637] As shown in Figure 30 and Table 23, BH01-BK58-MMAE and BH01-DK58-MMAE can significantly inhibit the growth of human colon cancer tumor cells HCT116 in a dose-dependent manner. At a dose of 3 mg / kg, the tumor inhibition rates of BH01-BK58-MMAE and BH01-DK58-MMAE on Day 14 were 91.34% and 95.52%, respectively. In addition, the effective dose of BH01-DK58-MMAE is relatively low, and it exhibits a tumor inhibition effect at a dose of 0.5 mg / kg (TGI% is 24.11%), indicating that BH01-DK58-MMAE has a large therapeutic window.

[0638] Table 23 Tumor inhibition rate of BH01-BK58-MMAE and BH01-DK58-MMAE on HCT116 xenograft tumors on Day 14

[0639] Example 36. Antitumor efficacy of BH01-DK58-MMAE and BH01-BK58-MMAE on NCI-H1975 xenograft tumors.

[0640] Experimental mice: Female B-NDG mice, 35 at the time of the experiment, 7 weeks old

[0641] Experimental cells: Human lung cancer tumor cells NCI-H1975 in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation;

[0642] Inoculum size: 5×10 6 / 100μL / mouse;

[0643] Inoculation site: right back area;

[0644] Grouped dosing: When the average tumor volume grows to 100-150mm 3 Around 24 hours, the tumor-bearing mice were randomly divided into groups and the drug was administered on the day of grouping, which was defined as Day 0. The drug was administered intravenously once a week for a total of 2 times. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. The tumor volume (mm 3 ) is calculated as (length × width 2) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0645] Dosage: 0.25mg / kg, 0.5mg / kg, 1mg / kg.

[0646] As shown in Figure 31 and Table 24, BH01-BK58-MMAE and BH01-DK58-MMAE can significantly inhibit the growth of human lung cancer tumor cells NCI-H1975, showing a dose-dependent relationship. On Day 17, both BH01-DK58-MMAE and BH01-BK58-MMAE showed significant tumor inhibition effects at a dose of 1 mg / kg (TGI% were 72.98% and 61.47%, respectively). In addition, the effective dose of BH01-DK58-MMAE is low, and it showed a tumor inhibition effect at a dose of 0.25 mg / kg (TGI% was 15.24%). At a dose of 0.5 mg / kg, it showed significant tumor inhibition compared to the control group on day 17.

[0647] Table 24 Tumor inhibition rate of BH01-BK58-MMAE and BH01-DK58-MMAE on NCI-H1975 xenograft tumors on Day 20

[0648] Example 37. C57BL / 6 mouse model has good tolerance to BH01-BK58-MMAE and BH01-DK58-MMAE

[0649] Experimental mice: C57BL / 6 mice, 6 weeks old, 48 females and 48 males.

[0650] Grouping and Dosing: Mice were randomly grouped according to body weight and dosed on the day of grouping, designated Day 0. Dosing was by intravenous injection once every two weeks for a total of two doses. Body weight was measured three times per week during the study, and the relationship between changes in mouse body weight and dosing time was recorded.

[0651] As shown in Figure 32 and Tables 25 and 26, the test results showed that the average body weight of animals in different dose groups and different genders increased, and there was no significant weight loss due to drug toxicity, indicating that C57BL / 6 mice tolerated BH01-BK58-MMAE and BH01-DK58-MMAE well at doses of 10 mg / kg and below.

[0652] Table 25 Body weight changes of C57BL / 6 mice injected intravenously with BH01-BK58-MMAE on Day 27

[0653] Table 26 Body weight changes of C57BL / 6 mice injected intravenously with BH01-DK58-MMAE on Day 28

[0654] Example 38. High-dose BH01-DK58-MMAE is less toxic to female and male C57BL / 6 mice than BH01-BK58-MMAE at the same dose

[0655] Experimental mice: C57BL / 6 mice, 6 weeks old, half male and half female (48 females and 48 males)

[0656] Group Dosing: Mice were randomly grouped according to body weight and dosed on the day of grouping, designated Day 0. Dosing was by intravenous injection, with a single dose. Body weight was measured three times per week during the study, and the relationship between changes in mouse body weight and dosing time was recorded.

[0657] As shown in Figure 33 and Tables 27 and 28, after intravenous injection of 12-20 mg / kg BH01-BK58-MMAE, female body weight changes on Day 8 ranged from 2.86% to 6.64%, with no significant differences observed. Male body weight changes ranged from -2.30% to 11.86%, with weight gains at 16 mg / kg and 20 mg / kg significantly lower than those at 12 mg / kg and PBS, indicating that BH01-BK58-MMAE is somewhat toxic to male mice. Following intravenous injection of 2-20 mg / kg BH01-DK58-MMAE, female body weight changes on Day 7 ranged from 1.97% to 5.37%, while male body weight changes ranged from 7.84% to 9.22%, with no significant differences observed, indicating that mice tolerate BH01-DK58-MMAE better.

[0658] The above results suggest that the strategy of controlling the affinity of the EGFR binding domain and truncating the hinge region to reduce EFGR targeting is beneficial to safety, significantly reducing toxicity to normal tissue cells, and making the prepared bispecific antibodies and ADCs have lower side effects.

[0659] Table 27 Body weight changes of C57BL / 6 mice injected intravenously with BH01-BK58-MMAE on Day 15

[0660] Table 28 Body weight changes of C57BL / 6 mice injected intravenously with BH01-DK58-MMAE on Day 14

[0661] Example 39. Internalization of BH01-DK58 Antibody is Stronger than Single-Target Antibodies and Benchmark Antibodies

[0662] method:

[0663] 1. Digest the target cells with Accutase, collect them and resuspend them in the assay buffer (corresponding complete culture medium).

[0664] 2. Adjust the target cell density to 1E5 cells / mL with assay buffer and transfer the cell suspension to the corresponding wells of a 96-well assay plate (Corning, Cat.#: 3595) (50 μL / well, 5000 cells / well).

[0665] 3. Place the experimental plate in a cell culture incubator (37°C / 5% CO2) and culture overnight to allow the target cells to adhere to the wall.

[0666] 4. Prepare the test / reference working solution (4×) and labeling reagent ( Human Fabfluor-pH Red Antibody Labeling Reagent, Sartorius, Cat.#: 4722) working solution (4×).

[0667] 5. Mix the test sample / reference sample working solution and the labeling reagent working solution in a 1:3 molar ratio and a 1:1 volume ratio, and incubate in a cell culture incubator (37°C / 5% CO2) for 15 minutes to allow the two to fully couple.

[0668] 6. According to the experimental plan, transfer the coupling mixture working solution to the corresponding wells of a 96-well experimental plate (50 μL / well).

[0669] 7. Place the test plate on The cells were incubated in an S3 live cell analyzer imaging system (37°C / 5% CO2). The imaging program (imaging channels: Phase and Red; objective lens: 10×; imaging interval: 2 hours; imaging cutoff: 48 hours) was set using the Incucyte software. The data processing system Controller took photos and images at the appropriate detection band and designated time points.

[0670] 8. Antibody internalization experimental data analysis (Incucyte method): The raw data and results of the experiment were processed by the live cell analyzer data processing system Live-Cell Analysis System analysis and export. Results are expressed as Total Red Object Area (μm² / Image). Exported raw data can be further analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6 software.

[0671] As shown in Figure 34 , the internalization effect of BH01-DK58 in HCT116 and NCI-H1975 cells was stronger than that of single-target antibodies KY301-01, KY303-58, and the corresponding antibodies Amivantamab, AZ-RAA / B09, and AZ-QD6 / B09.

[0672] Example 40. Protein levels of MET and EGFR in tumor cells

[0673] method:

[0674] 1. Prepare protein samples: Remove cells, place on ice, and centrifuge at 300 × g for 5 min at 4°C. Wash twice with pre-chilled PBS and remove the supernatant by centrifugation. Add RIPA lysis buffer to the cell pellet and shake thoroughly to lyse. Quantify protein using the BCA assay.

[0675] 2. Electrophoresis: Use a 4-12% precast gel, add 5 μL of protein marker and 18 μL of protein sample in sequence, plug in the power plug, adjust the voltage to 80-90 V, and after 15-30 minutes, adjust the voltage to 120-130 V when bromophenol blue enters the lower layer of gel. Stop electrophoresis when bromophenol blue just runs out of the gel.

[0676] 3. Transfer: Remove the PVDF membrane and use scissors to cut a piece of the same size and shape as the gel. Soak the cut PVDF membrane in methanol for 15 seconds before use to activate it. The activated PVDF membrane should completely cover the gel block. Remove the glass plate, cut the gel appropriately, and buckle the gel on the membrane. There should be no bubbles between the membrane and the gel block. Then cover the membrane with filter paper and sponge soaked in transfer buffer. Place the entire membrane in the transfer device and then in the transfer tank with the membrane side on the positive electrode and the gel side on the negative electrode. Place an ice box in the tank and fill it with transfer buffer. Adjust the voltage and set the appropriate transfer time according to the molecular weight of the target protein.

[0677] 4. Blocking: Wash the PVDF membrane 2-3 times with 0.1% TBST for 5 minutes each time, add the prepared 5% skim milk, and block on a shaker at room temperature for 60 minutes;

[0678] 5. Washing: Rinse the blocked PVDF membrane with 0.1% TBST three times, 5 minutes each time;

[0679] 6. Incubate with primary antibody: shake on a 4°C platform and block overnight;

[0680] 7. Washing: Rinse the blocked PVDF membrane with 0.1% TBST three times, 5 minutes each time;

[0681] 8. Incubate with secondary antibody: dilute HRP-labeled secondary antibody of the corresponding species in 0.1% TBST and incubate on a shaker at room temperature for 60 minutes;

[0682] 9. Washing: Rinse the NC membrane with 0.1% TBST three times, 10 minutes each time;

[0683] 10. Color development: Mix solution A and solution B in the ECL luminescent agent in a ratio of 1:1 and add them dropwise to the PVDF membrane. Expose, develop, fix and scan the film in a dark room.

[0684] As shown in Figure 35 , MET and EGFR proteins were expressed in nine cell types, including NCI-H1975, HCT116, HCC827, ASPC-1, EBC-1, BxPC-3, 786-O, A498, and HT29.

[0685] Example 41. KY-0301 (BH01-DK58-MMAE) downregulates MET and EGFR protein levels

[0686] method:

[0687] 1. Prepare protein samples: Remove cells, place on ice, and centrifuge at 300 × g for 5 min at 4°C. Wash twice with pre-chilled PBS and remove the supernatant by centrifugation. Add RIPA lysis buffer to the cell pellet and vortex thoroughly to lyse. Quantify protein using the BCA assay.

[0688] 2. Electrophoresis: Use a 4-12% precast gel, add 5 μL of protein marker and 18 μL of protein sample in sequence, plug in the power plug, adjust the voltage to 80-90 V, and after 15-30 minutes, adjust the voltage to 120-130 V when bromophenol blue enters the lower layer of gel. Stop electrophoresis when bromophenol blue just runs out of the gel.

[0689] 3. Transfer: Remove the PVDF membrane and use scissors to cut a piece of the same size and shape as the gel. Soak the cut PVDF membrane in methanol for 15 seconds before use to activate it. The activated PVDF membrane should completely cover the gel block. Remove the glass plate, cut the gel appropriately, and buckle the gel on the membrane. There should be no bubbles between the membrane and the gel block. Then cover the membrane with filter paper and sponge soaked in transfer buffer. Place the entire membrane in the transfer device and then in the transfer tank with the membrane side on the positive electrode and the gel side on the negative electrode. Place an ice box in the tank and fill it with transfer buffer. Adjust the voltage and set the appropriate transfer time according to the molecular weight of the target protein.

[0690] 4. Blocking: Wash the PVDF membrane 2-3 times with 0.1% TBST for 5 minutes each time, add the prepared 5% skim milk, and block on a shaker at room temperature for 60 minutes;

[0691] 5. Washing: Rinse the blocked PVDF membrane with 0.1% TBST three times, 5 minutes each time;

[0692] 6. Incubate with primary antibody: shake on a 4°C platform and block overnight;

[0693] 7. Washing: Rinse the blocked PVDF membrane with 0.1% TBST three times, 5 minutes each time;

[0694] 8. Incubate with secondary antibody: dilute HRP-labeled secondary antibody of the corresponding species in 0.1% TBST and incubate on a shaker at room temperature for 60 minutes;

[0695] 9. Washing: Rinse the NC membrane with 0.1% TBST three times, 10 minutes each time;

[0696] 10. Color development: Mix solution A and solution B in the ECL luminescent agent in a ratio of 1:1 and add them dropwise to the PVDF membrane. Expose, develop, fix and scan the film in a dark room.

[0697] As shown in Figure 36, after 24 hours of treatment of NCI-H1975, EBC-1, and HCC827 with 1 mg / mL KY-0301, the levels of MET and EGFR proteins were significantly decreased, and the phosphorylated MET and phosphorylated EGFR were reduced, indicating that KY-0301 can promote the endocytosis of MET and EGFR and inhibit the activation of MET and EGFR.

[0698] Example 42. KY-0301 (BH01-DK58-MMAE) is more effective in killing tumor cells than single-target-MMAE

[0699] method:

[0700] 1. Plate 786-O / ASPC-1 / A498 / BxPC-3 / EBC-1 / HCC827 / NCI-H1975 / HCT116 / HT29 cells into a black-walled, clear-bottom 96-well plate, using 100 μL of cell suspension per well.

[0701] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0702] 3. Add 100 μL CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and detect the fluorescence intensity

[0703] As shown in Figure 37, BH01-DK58-MMAE, benchmark antibodies AZD9592-MMAE, KY301-01-MMAE, and KY303-58-MMAE can all effectively kill tumor cells with medium, high, and low expression of MET and EGFR (786-O / ASPC-1 / A498 / BxPC-3 / EBC-1 / HCC827 / NCI-H1975 / HCT116 / HT29). Among them, the killing effect of BH01-DK58-MMAE is stronger than that of single-target ADCs (KY301-01-MMAE and KY303-58-MMAE), and is similar to the killing effect of AZD9592-MMAE (IC50 values ​​are shown in Table 29).

[0704] Table 29 IC50 values ​​of BH01-DK58-MMAE and AZD9592-MMAE in different tumor cells

[0705] Example 43. KY-0301 (BH01-DK58-MMAE) is less toxic to normal cells than the counterpart antibody AZD9592-MMAE

[0706] method:

[0707] 1. HFL-1 / MIHA / MRC-5 / BEAS-2B cells were plated into a black-walled, clear-bottomed 96-well plate, with 100 μL of cell suspension per well.

[0708] 2. Add 10 μL of serially diluted ADC to each well and incubate for 6 days;

[0709] 3. Add 100 μL CellTiter-Glo 2.0 Cell Viability Assay to each well, shake and mix for 10 minutes, and detect the fluorescence intensity

[0710] As shown in Figure 37, BH01-DK58-MMAE was toxic to HFL-1 / MRC-5 / BEAS-2B normal cells at a high concentration (20 μg / mL) and had little toxicity to MIHA cells. In contrast, the benchmark antibody AZD9592-MMAE killed HFL-1 / MRC-5 / BEAS-2B normal cells at a relatively low dose of 0.8 μg / mL and had a killing effect on MIHA cells at an extremely high dose of 100 μg / mL. These results indicate that BH01-DK58-MMAE is significantly less toxic to normal cells than the benchmark antibody AZD9592-MMAE, suggesting that BH01-DK58-MMAE is safer than AZD9592-MMAE.

[0711] Example 44. KY-0301 (BH01-DK58-MMAE) can cause cell cycle arrest in tumor cells

[0712] method:

[0713] 1. Digest HCT116 cells treated with BH01-DK58-MMAE for 48 hours to prepare a cell suspension;

[0714] 2. Take 50 μL of cell suspension, add 1 mL of pre-chilled PBS to resuspend the cells, resuspend, centrifuge, remove the supernatant, retain 50 μL of supernatant, and gently tap the bottom of the tube to disperse the cells;

[0715] 3. Add 1 mL of pre-cooled 70% ethanol, mix gently, fix at 4°C for more than 30 minutes, centrifuge, and retain the precipitate;

[0716] 4. Add 1 mL of pre-cooled PBS solution to resuspend and centrifuge;

[0717] 5. Add 0.5 mL of propidium iodide staining solution, slowly and thoroughly resuspend the cells, incubate at 37°C for 30 minutes, and store on ice.

[0718] 6. Flow cytometer detection.

[0719] As shown in Figure 39, after HCT116 cells were treated with BH01-DK58-MMAE for 48 h, the proportion of cells in the G2 / M phase was 43.76%, which was much higher than 18.8% in the blank control group, indicating that BH01-DK58-MMAE can induce G2 / M phase arrest in HCT116 cells.

[0720] Example 45. KY-0301 has significant antitumor efficacy against EBC-1 xenograft tumors

[0721] Experimental mice: female Balb / C Nude mice

[0722] Experimental cells: Human lung cancer tumor cells EBC-1 in the logarithmic growth phase were collected, the culture medium was removed and washed twice with PBS before inoculation;

[0723] Inoculum size: 5×10 6 / 100μL / mouse;

[0724] Inoculation site: right back area;

[0725] Grouped dosing: When the average tumor volume grows to 100-150mm 3Around 24 hours, the tumor-bearing mice were randomly divided into groups and the drug was administered on the day of grouping, which was defined as Day 0. The drug was administered intravenously once a week for 2 doses. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. The tumor volume (mm 3 ) is calculated as (length × width 2 ) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0726] Dosage: 1mg / kg, 2mg / kg, 4mg / kg.

[0727] KY-0301 is the BH01-DK58-MMAE described above, and its structural diagram is shown in Figure 40. As shown in Figure 41, KY-0301 significantly inhibited the growth of human lung cancer cells EBC-1 in a dose-dependent manner. On Day 17, the tumor inhibition rate of 4 mg / kg of KY-0301 was 100%.

[0728] Example 46. KY-0301 has significant antitumor efficacy against HT29 xenograft tumors

[0729] Experimental mice: female Balb / C Nude mice

[0730] Experimental cells: Human colorectal cancer HT29 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation;

[0731] Inoculum size: 5×10 6 / 100μL / mouse;

[0732] Inoculation site: right back area;

[0733] Grouped dosing: When the average tumor volume grows to 100-150mm 3 Around 24 hours, the tumor-bearing mice were randomly divided into groups and the drug was administered on the day of grouping, which was defined as Day 0. The drug was administered intravenously once a week for 2 doses. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. The tumor volume (mm 3 ) is calculated as (length × width 2 ) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0734] Dosage: 1mg / kg, 2mg / kg, 4mg / kg.

[0735] As shown in Figure 42, KY-0301 significantly inhibited the growth of human colorectal cancer cells HT29 in a dose-dependent manner. On Day 22, the tumor inhibition rate of KY-0301 at a dose of 4 mg / kg was 98%.

[0736] Example 47. KY-0301 has significant antitumor efficacy in the high tumor burden model and relapse model of NCI-H1975 xenograft tumors.

[0737] Experimental mice: female Balb / C Nude mice

[0738] Experimental cells: Human lung cancer tumor cells NCI-H1975 in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation;

[0739] Inoculum size: 5×10 6 / 100μL / mouse;

[0740] Inoculation site: right back area;

[0741] High tumor burden model group: When the average tumor volume of the PBS group grew to 3000mm 3 The tumor-bearing mice were randomly divided into groups and given 5 mg / kg of the drug twice on D26 and D33. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the time of administration was recorded. The tumor volume (mm 3 ) is calculated as (length × width 2 ) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0742] Recurrence model group: When the average tumor volume grows to 100-150mm 3 Around 24 hours after the start of treatment, tumor-bearing mice were randomly divided into groups and administered on the day of grouping, which was defined as Day 0. The drug was administered intravenously twice a week at a dose of 1.5 mg / kg. By Day 30, the tumor had resumed growth, and by Day 47, the tumor volume had grown to 1600 mm. 3 Around. 1.5 mg / kg was administered again on D47 and D54 to challenge the killing of KY-0301 on the regrowing tumor. During the experiment, the tumor volume and body weight were measured twice a week, and the relationship between the changes in the weight and tumor volume of the tumor-bearing mice and the administration time was recorded. The tumor volume (mm 3 ) is calculated as (length × width 2 ) / 2, tumor growth inhibition rate (TGI%) = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%;

[0743] As shown in Figure 43A, KY-0301 can significantly inhibit the growth of the human lung cancer NCI-H1975 high tumor burden model, with the tumor volume increasing from 3500 mm 3 Around until completely clear.

[0744] As shown in Figure 43B, KY-0301 can significantly inhibit the growth of the human lung cancer NCI-H1975 recurrence model, with the tumor volume increasing from 2000 mm 3 Around until completely clear.

[0745] Example 48. KY-0301 effectively inhibits tumor growth in a lung cancer PDX mouse model

[0746] 1. Prepare 45 female B-NDG mice (8-10 weeks old);

[0747] 2. Non-small cell lung cancer tumor tissue was cut into 2*2*2mm pieces and inoculated subcutaneously in the right anterior flank of B-NDG mice;

[0748] 3. Wait until the tumor grows to 200-300mm 3 The B-NDG mice were divided into 3 groups, with 6 mice in each group. The groups and drug administration are shown in the following table (Table 30):

[0749] Table 30 Animal grouping and drug administration

[0750] 4. PBS, KY-0301 1.5 mg / kg, and KY-0301 3 mg / kg were injected into the tail vein once a week for 2 weeks;

[0751] 5. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0752] 6. Calculation of tumor volume and tumor growth inhibition rate

[0753] Tumor volume (V) was calculated as: (length × width 2 ) / 2

[0754] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0755] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0756] The experimental results showed (Figure 44) that in the non-small cell lung cancer PDX B-NDG mouse model, KY-0301 doses of 1.5 mg / kg and 3 mg / kg were able to effectively inhibit the growth and proliferation of tumor tissue in mice, with the tumor inhibition rates on Day 24 being 79.5% and 97.9%, respectively, in a dose-dependent manner. The above results indicate that KY-0301 doses of 1.5 mg / kg and above can effectively inhibit the growth and proliferation of non-small cell lung cancer tissue in B-NDG mice in a dose-dependent manner.

[0757] Example 49. KY-0301 effectively inhibits tumor growth in a pancreatic cancer PDX mouse model

[0758] 1. Prepare 45 female B-NDG mice (8-10 weeks old);

[0759] 2. Pancreatic cancer tumor tissue was cut into 3*3*3mm pieces and inoculated subcutaneously in the right anterior flank of B-NDG mice;

[0760] 3. Wait until the tumor grows to 200-300mm 3 The B-NDG mice were divided into 3 groups, with 6 mice in each group. The groups and drug administration are shown in the following table (Table 31):

[0761] Table 31 Animal grouping and drug administration

[0762] 4. PBS, KY-0301 2 mg / kg, and KY-0301 4 mg / kg were administered by tail vein injection once a week for 2 weeks;

[0763] 5. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0764] 6. Calculation of tumor volume and tumor growth inhibition rate

[0765] Tumor volume (V) was calculated as: (length × width 2 ) / 2

[0766] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0767] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0768] The experimental results showed (Figure 45) that in the pancreatic cancer PDX B-NDG mouse model, KY-0301 at doses of 2 mg / kg and 4 mg / kg were able to effectively inhibit the growth and proliferation of tumor tissue in mice, with tumor inhibition rates of 93.7% and 98.8% on Day 32, respectively, showing a dose-dependent relationship. The above results indicate that KY-0301 at doses of 2 mg / kg and above can effectively inhibit the growth and proliferation of pancreatic cancer tissue in B-NDG mice in a dose-dependent manner.

[0769] Example 50. KY-0301 has a better tumor inhibition effect than the control product AZD9592-MMAE in the mouse HCT116 transplant tumor model

[0770] 1. Prepare 40 female Balb / c nude mice (4-5 weeks old);

[0771] 2. Human colon cancer HCT116 cells were cultured at 5×10 6 cells / cells were inoculated subcutaneously in the right anterior flank of mice;

[0772] 3. Wait until the tumor grows to 100-150mm 3 Balb / c nude mice were divided into 5 groups, with 5 mice in each group. The groups and dosing conditions are shown in the following table (Table 32):

[0773] Table 32 Animal grouping and drug administration

[0774] 4. PBS, KY-0301 1.5 mg / kg, KY-0301 3 mg / kg, AZD9592-MMAE 2.8 mg / kg, and AZD9592-MMAE 5.6 mg / kg were administered via tail vein injection once a week for 2 weeks.

[0775] 5. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0776] 6. Calculation of tumor volume and tumor growth inhibition rate

[0777] Tumor volume (V) was calculated as: (length × width 2 ) / 2

[0778] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0779] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0780] Experimental results (Figure 46) show that in a Balb / c nude mouse model bearing HCT116 colon cancer xenografts, KY-0301 at doses of 1.5 mg / kg and 3 mg / kg, and AZD9592-MMAE at doses of 2.8 mg / kg and 5.6 mg / kg, effectively inhibited tumor cell growth and proliferation in mice, with Day 17 inhibition rates of 41.3%, 67.0%, 32.2%, and 45.7%, respectively. KY-0301 and AZD9592-MMAE exhibited dose-dependent responses. The molar dose of KY-0301 (DAR=4) 1.5 mg / kg is equal to the molar dose of AZD9592-MMAE (DAR=4) 2.8 mg / kg, the molar dose of KY-0301 (DAR=4) 3 mg / kg is equal to the molar dose of AZD9592-MMAE (DAR=4) 5.6 mg / kg, the tumor inhibition rate of low-dose KY-0301 is higher than that of low-dose AZD9592-MMAE (41.3%>32.2%), and the tumor inhibition rate of high-dose KY-0301 is higher than that of high-dose AZD9592-MMAE (67.0%>45.7%).

[0781] The above results show that KY-0301 at doses of 1.5 mg / kg and above can effectively inhibit the growth and proliferation of colon cancer cells in mice in a dose-dependent manner; at equimolar doses and equal payloads, KY-0301 has a better tumor inhibition effect than AZD9592-MMAE.

[0782] Example 51. KY-0301 has a better tumor inhibition effect than the benchmark product AZD9592-Dxd in the mouse EBC-1 xenograft tumor model

[0783] 1. Prepare 40 female Balb / c nude mice (4-5 weeks old);

[0784] 2. Human non-small cell lung cancer cells were cultured at 5×10 6 cells / cells were inoculated subcutaneously in the right anterior flank of mice;

[0785] 3. Wait until the tumor grows to 100-200mm 3 Balb / c nude mice were divided into 5 groups, with 5 mice in each group. The groups and dosing conditions are shown in the following table (Table 33):

[0786] Table 33 Animal grouping and drug administration

[0787] 4. PBS, KY-0301 1 mg / kg, KY-0301 2 mg / kg, KY-0301 3 mg / kg, and AZD9592-DXd 3.7 mg / kg were administered via tail vein injection once a week for 2 weeks.

[0788] 5. Measure the tumor volume and body weight twice a week, and record the relationship between the changes in body weight and tumor volume of tumor-bearing mice and the administration time;

[0789] 6. Calculation of tumor volume and tumor growth inhibition rate

[0790] Tumor volume (V) was calculated as: (length × width 2 ) / 2

[0791] The tumor growth inhibition rate (TGI%) was calculated using the following formula:

[0792] Tumor growth inhibition rate = (1-tumor volume change in drug-treated group / tumor volume change in control group) × 100%

[0793] The experimental results showed ( FIG. 47 ) that, in the Balb / c nude mouse model of EBC-1 lung cancer xenografts, KY-0301 at doses of 1 mg / kg, 2 mg / kg, and 3 mg / kg, and AZD9592-DXd at 3.7 mg / kg, all effectively inhibited the growth and proliferation of tumor cells in mice. The tumor inhibition rates on Day 14 were 66.3%, 97.3%, 100.0%, and 96.3%, respectively. The inhibitory effect of KY-0301 on tumors was dose-dependent. The molar dose of 2 mg / kg of KY-0301 (DAR=4) was equivalent to the molar dose of 3.7 mg / kg of AZD9592-DXd (DAR=6). At equimolar doses, the average tumor volume in the 2 mg / kg group of KY-0301 (DAR=4) on Day 20 was 116.0 mm. 3 The tumor volume increased by -35.8% compared with the initial tumor volume. The average tumor volume in the AZD9592-DXd (DAR=6) 3.7 mg / kg group was 221.5 mm 3 , an increase of 22.0% compared with the initial tumor volume; the above results show that KY-03011mg / kg and above can effectively inhibit the growth and proliferation of non-small cell lung cancer cells in mice in a dose-dependent manner; at equimolar doses, KY-0301 has a better tumor inhibition effect than AZD9592-DXd.

[0794] Example 52. KY-0301 showed good safety in primate pre-toxicology experiments.

[0795] Experimental animals: 4 rhesus monkeys, half male and half female

[0796] Group administration: Four rhesus monkeys were randomly divided into two groups (two monkeys in each group, half male and half female), including low-dose (1 / 6 mg / kg, the dose was increased from D29) and high-dose (3 mg / kg) KY-0301 groups. The low-dose and high-dose groups were administered once on D1 and D15, with doses of 1 mg / kg and 3 mg / kg, respectively. The low-dose group increased the dose on D29 (adjusted from 1 mg / kg to 6 mg / kg) and was administered once on D29 and D43.

[0797] Results: No unplanned deaths were observed during the study. Clinical observations, body weight, food intake, coagulation, serum biochemistry, and immune function tests revealed no significant KY-0301-related abnormalities. KY-0301-related abnormalities were primarily: At doses ≥3 mg / kg, decreases in RBC, HGB, HCT, #RET, RET%, #NEU, #LYMP, and / or #MONO were observed in both males and females one week after dosing (D7, D35, or D49). In addition, increases in WBC, #LYMP, and #MONO were observed in both males and females two weeks after dosing (D14 or D42). All of these abnormalities resolved or showed signs of resolution by the end of the observation period (D71). The highest non-severely toxic dose (HNSTD) of KY-0301 administered intravenously was 6 mg / kg in both males and females. After administration on D1 (or D29), there were no gender differences in ADC, total anti-drug exposure (measured in AUC(0-t) and Cmax) within the dose range of 1-6 mg / kg.

[0798] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A nanobody targeting MET, characterized in that: The VHH chain of the Nanobody has the following complementarity determining regions CDR1, CDR2 and CDR3: CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:

15.

2. The Nanobody according to claim 1, characterized in that The VHH chain of the MET-targeting Nanobody has an amino acid sequence with an homology of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% to the amino acid sequence shown in SEQ ID NO: 1, or has an amino acid sequence as shown in SEQ ID NO:

1.

3. An antibody targeting MET, characterized in that: The antibody comprises one or more VHH chains of the MET-targeting Nanobody according to claim 1.

4. A nanobody targeting EGFR, characterized in that: The VHH chain of the Nanobody has the following complementarity determining regions CDR1, CDR2 and CDR3: CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO:

18.

5. The Nanobody according to claim 4, characterized in that The VHH chain of the EGFR-targeting Nanobody has an amino acid sequence with an homology of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% to the amino acid sequence shown in SEQ ID NO: 3, or has an amino acid sequence as shown in SEQ ID NO:

3.

6. An antibody targeting EGFR, characterized in that: The antibody comprises one or more VHH chains of the EGFR-targeting Nanobody as claimed in claim 4.

7. A multispecific antibody comprising the VHH chain of the MET-targeting Nanobody as claimed in claim 1 and the VHH chain of the EGFR-targeting Nanobody as claimed in claim 4.

8. The multispecific antibody according to claim 7, characterized in that The multispecific antibody is a bispecific antibody targeting MET and EGFR, and has a structure shown in Formula II from the N-terminus to the C-terminus: in, V1, V2, V3, V4, V5, and V6 are each independently none, a VHH chain of the Nanobody targeting MET, or a VHH chain of the Nanobody targeting EGFR, At least one of V1, V2, and V3 is a VHH chain of a nanobody targeting MET, and at least one of V4, V5, and V6 is a VHH chain of a nanobody targeting EGFR; L1, L2, L3, L4, L5, and L6 are each independently none or a connecting peptide; H1 and H2 are each independently free or an immunoglobulin hinge region; Fc1 and Fc2 are each independently an immunoglobulin Fc segment; "‖" represents a disulfide bond or a knob-into-hole connection; "-" represents a peptide bond.

9. The multispecific antibody according to claim 8, wherein The bispecific antibody has a structure shown in the following formula (IIa) from N-terminus to C-terminus: in, One of V1 and V2 is the VHH chain of a nanobody targeting MET, and the other is the VHH chain of a nanobody without or targeting EGFR; One of V4 and V5 is the VHH chain of a nanobody targeting EGFR, and the other is the VHH chain of a nanobody without or targeting MET; The remaining elements are as defined in formula (II).

10. The multispecific antibody according to claim 8, wherein It comprises a first antigen-binding chain and a second antigen-binding chain, wherein The amino acid sequence of the first antigen-binding chain is shown in any one of SEQ ID NO: 2, 6, 11, 12; and / or The amino acid sequence of the second antigen-binding chain is shown in any one of SEQ ID NOs: 5, 7-12.

11. The multispecific antibody according to claim 10, characterized in that The bispecific antibody comprises a first antigen binding chain shown in SEQ ID NO: 2 and / or a second antigen binding chain shown in SEQ ID NO: 5 or 7.

12. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, which is a Nanobody targeting MET as described in claim 1, an antibody targeting MET as described in claim 3, a Nanobody targeting EGFR as described in claim 4, an antibody targeting EGFR as described in claim 6, or a multispecific antibody as described in claim 7; and (b) a coupling moiety coupled to said Nanobody portion, said coupling moiety being selected from the group consisting of a detectable label, a drug, or a combination thereof.

13. The immunoconjugate according to claim 12, characterized in that The coupling moiety is MMAE.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) a Nanobody targeting MET as described in claim 1, an antibody targeting MET as described in claim 3, a Nanobody targeting EGFR as described in claim 4, an antibody targeting EGFR as described in claim 6, a multispecific antibody as described in any one of claims 7 to 11, or an immunoconjugate as described in claim 14; (ii) a pharmaceutically acceptable carrier.

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