Patent Application
20240165253
The present disclosure relates to the field of antibody-drug conjugates, and specifically relates to an antibody-drug conjugate targeting c-Met and applications thereof.
c-Met (tyrosine-protein kinase Met) is a proto-oncogene (Reference 1: Edakuni G, Sasatomi E, Satoh T, et al. Expression of the hepatocyte growth factor/c-Met pathway is increased at the cancer front in breast carcinoma. [J]. Pathology International, 2010, 51 (3): 172-178), and the post-transcriptional product is hepatocyte growth factor (HGF) receptor. Consistent with HGF, its precursor is a single chain, which is broken into a chain and β chain connected by disulfide bonds after various modifications. Cellularly, c-Met is mainly divided into intracellular and extracellular parts. For β chain, the extracellular region includes SD (Sema domain), PSID (plexin-semaphorin-integrin domain) and IPTD (immunoglobulin-like regions in plexins and transcription factors domain), and the intracellular region mainly includes JD (juxtamembrane domain) and TKCD (a tyrosine kinase domain (TK domain) and a c-terminal docking site) (Reference 2: Sattler M, Reddy M, Hasina R, et al. The role of the c-Met pathway in lung cancer and the potential for targeted therapy [J]. Therapeutic Advances in Medical Oncology, 2011, 3(4):171-84.). c-Met is the receptor of HGF. When HGF binds to c-Met, the structure of c-Met is changed, which activates the intracellular protein tyrosine kinase, resulting in the phosphorylation of itself, mainly the phosphorylation of Try1235 and Try1234. Upon activation of c-Met, Try1349 and Try1256 at the end of the β chain are phosphorylated (Reference 3: Peruzzi B, Bottaro D P. Targeting the c-Met Signaling Pathway in Cancer [J]. Clinical Cancer Research, 2006, 12(12):3657-3660). When HGF binds to c-Met, multiple signaling pathways are triggered: (1) Ras-Rac signaling pathway leads to the movement of the cytoskeleton; (2) Ras-MAPK cascade promotes cell division and differentiation; (3) PI3K-FAK signaling pathway promotes cell migration and invasion; and (4) PI3K-AKT signaling pathway promotes cell survival. The HGF/c-Met signaling pathway promotes growth and migration of tumor cells.
c-Met is overexpressed on many tumor cells, mainly head and neck squamous carcinoma and hypopharyngeal carcinoma, but also on renal cell carcinoma, colorectal cancer, lung adenocarcinoma, ovarian cancer, liver cancer, breast cancer, bladder cancer, non-small cell carcinoma and gastric cancer. The high expression in tumor cells and low expression in normal cells of c-Met makes c-Met an excellent target for targeted therapy.
Antibody-drug conjugate (ADC) is a biological drug that links a biologically active drug and an antibody through a chemical linker. In recent years, a number of antibody-drug conjugates have made breakthroughs in the treatment of malignant tumors, making them a major emerging treatment method after surgery, chemotherapy and radiotherapy. However, as of March 2021, only 11 antibody conjugated drugs have been approved in the world (10 have been approved by the US FDA, and one has been approved by the Japanese PMDA), and there are only a few approved indications, which are far from meeting the current clinical needs of patients with malignant tumors.
At present, the biological drugs targeting c-Met in the research or clinical stage are mainly monoclonal antibodies, and there are few antibody-drug conjugates (ADC) (Reference 4: LILIANE GOETSCH, A. J. Anti-CMET antibody and its use for the detection and the diagnosis of cancer. WO2011020925 A1, 2012). Among them, SHR-A1403 for injection developed by Hengrui Medicine is an ADC formed by chemical coupling of a humanized anti-c-Met monoclonal antibody and a microtubule inhibitor. By binding to c-Met on the surface of tumor cells, this antibody-drug conjugate can be endocytosed into tumor cells, and then release small molecule toxins after degradation in lysosome, which plays a role in killing tumor cells (Reference 5: Ki-Hyun Kim Progress of antibody-based inhibitors of the HGF-cMET axis in cancer therapy, Experimental & Molecular Medicine (2017) 49, e307). SHR-A1403 was approved by the FDA for clinical trials in the United States in January 2017. According to the FDA official website (https://clinicaltrials.gov/ct2/show/NCT03856541?term=SHR-A1403&rank=1), the Phase I clinical trial is currently in the process of recruiting patients.
ABBVie's ABBV-399 is also in phase I clinical research in the United States. It is a conjugate of ABT-700 antibody and MMAE with a DAR (drug-to-antibody ratio) of 3.1 for the treatment of c-Met-overexpressing non-small cell lung cancer. The results of early clinical trials of c-Met showed that ABBV-399 can produce cytotoxic effect on c-Met positive cancer cells. The phase I clinical trial (NCT02099058) was conducted in 29 patients with c-Met-positive NSCLC, and the safety and efficacy of ABBV-399 alone or in combination with erlotinib were preliminarily investigated. In the single administration cohort, >10% of patients experienced adverse reactions, mainly fatigue, nausea, neuralgia, and nausea (43.8%, 37.5%, 25%, and 18.8%, respectively). In the single administration group, 3/16 patients experienced partial remission, lasting between 3.1 and 11.1 months. After 12 weeks of administration, 56.3% of patients in the single administration group were under control. In the combined administration group, >10% of patients also experienced adverse reactions, mainly neuralgia and nausea (46.2 and 23.1%, respectively). Partial remission occurred in 4/13 patients in this group, of which 1 remains to be confirmed, lasting between 2.8 to 9.1 months. After 12 weeks of treatment, 76.9% of patients in the combined administration group were under control. There were no treatment-related deaths in either the single administration group or the combined administration group. The trial results confirmed that ABBV-399 was well tolerated at a dose of 2.7 mg/kg, and showed good anti-tumor activity in HGF-positive NSCLC patients (Reference 6: Wang J, Anderson M G, Oleksijew A, et al. al. ABBV-399, a c-Met Antibody-drug conjugate that Targets Both MET Amplified and c-Met Overexpressing Tumors, Irrespective of MET Pathway Dependence [J]. Clinical Cancer Research, 2016, 23(4):992-1000.). According to the latest data released by ABBVie in 2016, about 19% of NSCLC patients with H-score ≥150 experienced partial remission after treatment with ABBV-399. The tolerated dose of ABBV-399 was 2.7 mg/kg every three weeks, and the most frequently occurring toxicities were fatigue (25%), nausea (23%), neuralgia (15%), decreased appetite (13%), vomiting (13%), and diarrhea (10%) (Reference 7: Angevin E, Kelly K, Heist R, et al. First-in-human phase 1, dose-escalation and -expansion study of ABBV-399, an antibody-drug conjugate (ADC) targeting c-Met, in patients (pts) with advanced solid tumors [J]. Annals of Oncology, 2016, 27(suppl 6).
Although dozens of antibodies targeting c-Met are currently in clinical stage, the tumor curative effect is not satisfactory. ADC drugs can effectively combine these well-targeted monoclonal antibodies and cytotoxins to maintain targeting while enhancing the killing effect on cells. Currently, the development of ADCs targeting c-Met is relatively slow, few in the clinical stage, and they are in the early clinical stage, such as ABBVie's ABBV-399 and Hengrui Medicine's SHR-A1403, both of which are in phase I clinical trials. The safety of drugs and effective targeted indications are still under exploration. Therefore, there is an urgent need to develop anti-c-Met antibody-drug conjugates with better efficacy and safety in clinical practice, and more targeted indications can be expanded for effective treatment.
The present disclosure provides an antibody targeting c-Met or antigen-binding fragment thereof and use thereof in the treatment of cancer. The present disclosure also provides an antibody-drug conjugate (ADC) including the above-mentioned antibody, a nucleotide encoding the above-mentioned c-Met antibody, a polynucleotide combination, an expression vector and a host cell, a pharmaceutical composition including the above-mentioned antibody targeting c-Met or antibody-drug conjugate, as well as their use in the manufacture of a medicament for the treatment or prevention of a cancer.
Specifically, the present disclosure provides a humanized antibody targeting c-Met or antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, and:
(1) the heavy chain variable region has a sequence shown in SEQ ID NO: 7, or a sequence that has the same CDRs 1-3 as SEQ ID NO: 7 and an identity of greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 7; and/or
(2) the light chain variable region has a sequence shown in SEQ ID NO: 8, or a sequence that has the same CDRs 1-3 as SEQ ID NO: 8 and an identity of greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 8.
In some embodiments, the humanized antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibody, bispecific antibody, multispecific antibody, recombinant protein including the antigen-binding fragment, Fab 5 fragment, F(ab′) fragment, F(ab′) 2 fragment, FIT fragment, dAb, Fd, and single chain antibody (scFv).
In some embodiments, the antibody or antigen-binding fragment further comprises a constant region of an immunoglobulin, and the immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.
In some specific embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence shown in SEQ ID NO: 7; and/or the light chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence shown in SEQ ID NO: 8.
The amino acid sequence shown in SEQ ID NO: 7 is as follows:
The amino acid sequence shown in SEQ ID NO: 8 is as follows:
In other specific embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence shown in SEQ ID NO: 9; and/or the light chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence shown in SEQ ID NO: 10.
The amino acid sequence shown in SEQ ID NO: 9 is as follows:
The amino acid sequence shown in SEQ ID NO: 10 is as follows:
The present disclosure also relates to an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of the above.
The present disclosure also relates to a nucleic acid construct including the above-described polynucleotide.
The above-described nucleic acid construct is preferably an expression vector, and the polynucleotide is operably linked to a regulatory sequence that permits expression of a polypeptide encoded by the polynucleotide in a host cell or cell-free expression system.
The present disclosure also relates to a host cell including the above-described polynucleotide or expression vector, and the host cell is preferably selected from the group consisting of a prokaryotic cell, eukaryotic cell, yeast cell, mammalian cell and E. coli cell; and further, the host cell is preferably selected from the group consisting of a CHO cell, NSO cell, Sp2/0 cell and BHK cell.
The present disclosure also provides a method for producing the antibody or antigen-binding fragment thereof described in any one of the above, the method including culturing the above-described host cell under a condition allowing the expression of the above-described nucleic acid construct, and recovering the produced expressed protein from the culture.
The present disclosure also provides an antibody-drug conjugate targeting c-Met, which is a conjugate of any one of the above-mentioned antibody or antigen-binding fragment thereof and one or more therapeutic agents.
In some embodiments, the therapeutic agent is selected from the group consisting of a cytotoxic molecule, immunoenhancer and radioisotope, the cytotoxic molecule includes but is not limited to a tubulin inhibitor or DNA damaging agent; further preferably, the tubulin inhibitor includes but is not limited to a cytotoxic molecule of dolastatins and auristatins and a cytotoxic molecule of maytansines; the DNA damaging agent includes but is not limited to calicheamicins, duocarmycins, pyrrolobenzodiazepine derivatives (PBD), camptothecins and derivatives thereof, SN-38 and Dxd; more preferably, the cytotoxic molecule of auristatins includes but is not limited to MMAE, MMAF, and a derivative thereof, and the cytotoxic molecule of maytansines includes but is not limited to DM1, DM4, and a derivative thereof.
The preferred general structural formula of the antibody-drug conjugate provided by the present disclosure is A-(L-U)n, and: A represents the antibody or antigen-binding fragment thereof described in any one of the above; U is an active drug unit; L is any linking group, and is covalently linked to the antibody or antigen-binding fragment A and the active drug unit U, respectively; n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8; and A is linked to 1, 2, 3, 4, 5, 6, 7 or 8 of the active drug unit U through one or more of the linking group L.
The linking group L is covalently linked to the amino residue or thiol residue on the antibody targeting c-Met or antigen-binding fragment A; preferably, the linking group L is covalently linked to the thiol residue on the antibody targeting c-Met or antigen-binding fragment A; more preferably, the linking group L is covalently linked to the thiol residue formed after the interchain disulfide bond on the antibody targeting c-Met or antigen-binding fragment A is opened. The linking group L includes a cleavable linker and a non-cleavable linker. The cleavable linker includes a peptide unit including 2-20 amino acids, and preferably, the peptide unit is selected from the group consisting of -valine-citrulline- (-Val-Cit-), -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylalanine-lysine- (-Phe-Lys-), -phenylalanine-arginine- (-Phe-Arg-) and a combination thereof.
In some embodiments, L includes a structure selected from the group consisting of:
In some embodiments, the active drug unit U has a structure selected from the group consisting of:
In some embodiments, the antibody-drug conjugate has a structure selected from the group consisting of:
wherein, n in ADC-1 is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8; or
wherein, n in ADC-2 is an integer selected from 1, 2, 3 and 4; or
wherein, n in ADC-3 is an integer selected from 1, 2, 3 and 4; or
wherein, n in ADC-4 is an integer selected from 1 and 2; or
wherein, n in ADC-5 is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8; or
wherein, n in ADC-6 is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8; or
and, n in ADC-7 is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8.
The present disclosure also provides a pharmaceutical composition including the antibody or antigen-binding fragment thereof and/or antibody-drug conjugate described in any one of the above, and a pharmaceutically acceptable carrier.
The present disclosure also provides use of any one of the above-described antibody or antigen-binding fragment thereof, polynucleotide, nucleic acid construct, expression vector, antibody-drug conjugate or pharmaceutical composition in the manufacture of a medicament for treating or preventing a cancer.
In some embodiments, the cancer is a c-Met positive cancer.
In some embodiments, the c-Met positive cancer includes lung cancer and gastric cancer.
The present disclosure also provides a method of treating a disease, including administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate or pharmaceutical composition described in any one of the above; and the disease is a c-Met positive cancer, preferably lung cancer and gastric cancer.
The present disclosure also provides a kit including a container, a preparation placed in the container and an instruction; and the preparation comprises the antibody, antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition described in any one of the above.
The present disclosure also provides use of the antibody or antigen-binding fragment thereof, antibody-drug conjugate, polynucleotide, nucleic acid construct, expression vector, or pharmaceutical composition described in any of the above in the manufacture of a medicament for treating or preventing a cancer. In one embodiment, the cancer is a solid tumor; and the solid tumor further includes lung cancer and gastric cancer.
The present disclosure also provides a recombinant protein including the antibody or antigen-binding fragment described in any one of the above.
The above-mentioned recombinant protein is preferably a bispecific antibody or multispecific antibody, and the multispecific antibody is preferably trispecific antibody, tetraspecific antibody, pentaspecific antibody, etc.
The present disclosure also provides use of any one of the above-mentioned antibody or antigen-binding fragment in the preparation of a recombinant protein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those of ordinary skill in the art. For definitions and terms in the art, those skilled in the art can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Although the numerical ranges and parameter approximations are shown in the broad scope of the present disclosure, the numerical values shown in the specific examples are recorded as accurately as possible. However, any numerical value must inherently include an error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood as covering any and all subranges therein. For example, a recorded range of “1 to 10” should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (inclusive); that is, all sub-ranges beginning with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as “incorporated herein” should be understood as being incorporated in its entirety.
It should also be noted that, as used in this specification, the singular form includes the plural form of the object to which it refers, unless clearly and explicitly limited to one object to which it refers. The term “or” may be used interchangeably with the term “and/or” unless the context clearly indicates otherwise.
As used herein, the terms “pharmaceutical composition”, “combined drug”, or “pharmaceutical combination” are used interchangeably, and means a combination of at least one drug and may have a pharmaceutically acceptable carrier or adjuvant material combined together to achieve a particular purpose. In one embodiment, the pharmaceutical composition comprises a combination that is separated in time and/or space as long as it can work together to achieve embodiments of the present disclosure. For example, the components contained in the pharmaceutical composition (for example, the antibody, nucleic acid molecule, nucleic acid molecule combination, and/or antibody-drug conjugate according to the present disclosure) may be administered to a subject as a whole or separately. When the ingredients contained in the pharmaceutical composition are separately administered to a subject, the ingredients can be administered to the subject simultaneously or sequentially. In one embodiment, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycol such as polypropylene glycol, triglyceride and the like. The type of the pharmaceutically acceptable carrier used depends, inter alia, on whether the composition according to the present disclosure is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present disclosure may contain a wetting agent, an emulsifier or a buffer substance as an additive.
The pharmaceutical composition, vaccine or pharmaceutical formulation according to the present disclosure can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
The term “therapeutic agent” as used herein refers to any substance or entity plays a therapeutic role (e.g., treating, preventing, relieving or inhibiting any disease and/or condition), including but not limited to a chemotherapeutic agent, radiotherapy agent, immunotherapeutic agent, thermally therapeutic agent.
As used herein, “CDR region” or “CDR” refers to the hypervariable region of the heavy and light chain of an immunoglobulin, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy chain CDRs and three light chain CDRs. Depending on the circumstances, the term CDR or CDRs used herein is used to indicate one of these regions, or several or even all of these regions, which region contains most of the amino acid residues responsible for binding based on the affinity of an antibody for an antigen or its recognition epitope.
For the present disclosure, “consistency”, “identity” or “similarity” between two nucleic acid or amino acid sequences refers to the percentage of the same nucleotide or amino acid residues between the two sequences to be compared obtained after the optimal alignment. The percentage is purely statistical and the differences between the two sequences are randomly distributed and cover their full length. Comparisons between two nucleic acid or amino acid sequences are usually performed by comparing these sequences after aligning them in an optimal manner, and can be performed over a segment or over a “comparison window”. In addition to manual implementation, the optimal alignment used for comparing sequences can also be performed by the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by the local homology algorithm of Neddleman and Wunsch (1970) [J. MoI. Biol. 48:443], by the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), or by a computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by a comparison software BLAST N or BLAST P).
As used herein, “therapeutically effective amount” or “effective amount” refers to a dose sufficient to demonstrate a benefit when administered to a subject. The actual amount administered, as well as the frequency and time course of administration will depend on the condition and severity of the subject to be treated. General practitioners and other doctors are ultimately responsible for the treatment prescription (for example, decision on the dose) and usually make decisions based on the disease being treated, the condition of the individual patient, the delivery site, the mode of administration, and other factors known to the doctor.
The term “subject” as used herein refers to a mammal, such as human, but may also refer to other animals, such as wild animals (such as heron, stork, crane), domestic animals (such as duck, goose) or experimental animals (such as orangutan, monkey, rat, mouse, rabbit, guinea pig, marmot, ground squirrel).
The term “antibody” includes monoclonal antibody, bispecific antibody, multispecific antibody or recombinant protein including antigen-binding fragment; the term “antigen-binding fragment” includes Fab fragment, F(ab′) fragment, F(ab′)2 fragment, Fv fragment, dAb, Fd, single chain antibody (scFv), scFv-Fc fragment or diabody, or any fragment that should be able to increase half-life by chemical modification or by incorporation into liposomes, such chemical modifications as the addition of poly(alkylene) glycols such as polyethylene glycol (“PEGylation”) (PEGylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)2-PEG or Fab′-PEG) (“PEG” refers to polyethylene glycol), and such fragment has EGFR binding activity. In one embodiment, the functional fragment will consist of or comprise partial sequences of the heavy or light variable chain of the antibody from which it is derived, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably for EGFR at least equal to 1/100 of the affinity of the antibody from which it is derived, more preferably at least equal to 1/10. Such a functional fragment comprises a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which it is derived.
The term “humanized antibody” refers to an antibody including a CDR region derived from a non-human antibody, and the other portion of the antibody is derived from one (or several) human antibody (antibodies). Moreover, in order to retain binding affinity, some residues of the backbone (referred to as FR) segment may be modified (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). A humanized antibody or fragment thereof according to the present disclosure can be prepared by known techniques;
The term “chimeric antibody” refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. The chimeric antibody or fragment thereof according to the present disclosure can be prepared by using genetic recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA including a promoter and a sequence encoding the variable region of the non-human and especially murine monoclonal antibody according to the present disclosure, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present disclosure encoded by this recombinant gene will be, for example, a murine-human chimera, and the specificity of the antibody is determined by the variable region derived from murine DNA, and its isotype is determined by the constant region derived from human DNA. Method for preparing chimeric antibodies can be found in, for example, Verhoeyn et al. (BioEssays, 8:74, 1988).
The term “monoclonal antibody” refers to a preparation of antibody molecules having a single molecular composition. The monoclonal antibody composition shows a single binding specificity and affinity for a specific epitope.
The term “AAJ8D6 antibody” used herein, unless otherwise specified, refers to the humanized monoclonal antibody AAJ8D6 targeting c-Met obtained by the present disclosure.
The cAAJ8D6 antibody used herein refers to a human-murine chimeric antibody including a murine variable region and a human constant region. The difference between the cAAJ8D6 antibody and the AAJ8D6 antibody is only in the framework region in the variable region that the framework region of cAAJ8D6 is of murine origin, and the framework region of AAJ8D6 is of human origin.
In general, in order to prepare a monoclonal antibody or functional fragment thereof, especially of murine origin, reference may be made to the techniques described in particular in the manual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256:495-497, 1975).
The monoclonal antibody according to the present disclosure can be purified, for example, on an affinity column on which c-Met antigen has been immobilized in advance or which comprises one of the fragments of the epitope that can be specifically recognized by the monoclonal antibody according to the present disclosure. More specifically, the monoclonal antibody can be purified by protein A and/or G chromatography, in the presence or absence of ion exchange chromatography aimed at eliminating residual protein contaminants as well as DNA and LPS with or without size exclusion chromatography on Sepharose gels aimed at eliminating potential aggregates due to the presence of dimers or other multimers. More preferable, all of these techniques can be used simultaneously or sequentially.
The term “dolastatin” as used herein refers to a polypeptide isolated from a marine organism, Dollabella auricularia, which includes, but is not limited to, dolastatin 10 and dolastatin 15. Dolastatin is a mitotic inhibitor that exhibits strong anticancer activity, and is therefore a candidate for anticancer drugs. The researchers further discovered and synthesized many derivatives of dolastatin, such as MMAE and MMAF.
The term “linker” or “linking group” as used herein refers to the moiety of an antibody-drug conjugate (i.e. ADC) that links an antibody to a drug, which may or may not be cleavable. Cleavable linkers (i.e., breakable linkers or biodegradable linkers) can be cleaved in or on target cells to release the drug. In one embodiment, the linker of the present disclosure has very good stability, greatly reducing the release of the drug before delivery to the target (e.g., in the blood), to reduce side effects and toxicity. In some specific embodiments, the linker of the present disclosure is selected from cleavable linkers, such as disulfide-based linkers (which are selectively cleaved in tumor cells with higher thiol concentrations), peptide linkers (which are cleaved by enzymes in tumor cells) and hydrazone linker. In other specific embodiments, the linker of the present disclosure is selected from non-cleavable linkers, such as thioether linkers. In one embodiment, the linker of the present disclosure is selected from the group consisting of cleavable mc-vc-PAB linker and non-cleavable mc linker.
Based on the c-Met murine antibody mAAJ8D6, a variety of chimeric antibodies and humanized antibodies were further designed and compared for affinity, and the candidate chimeric c-Met antibody cAAJ8D6 and humanized anti-antibody AAJ8D6 were finally determined.
Amino acid sequence of the heavy chain variable region of anti-c-Met humanized antibody AAJ8D6 (SEQ ID NO: 7):
Amino acid sequence of the light chain variable region of anti-c-Met humanized antibody AAJ8D6 (SEQ ID NO: 8):
Amino acid sequence of the heavy chain of anti-c-Met humanized antibody AAJ8D6 (SEQ ID NO: 9):
Amino acid sequence of the light chain of anti-c-Met humanized antibody AAJ8D6 (SEQ ID NO: 10):
The affinities of chimeric cAAJ8D6 and the humanized antibody AAJ8D6 were determined by ELISA. A 96-well plate was coated with soluble ED protein and then incubated with diluted antibodies. ED-related antibodies were detected with an HRP-conjugated goat F(ab′)2 anti-human IgG Fc-specific secondary antibody. The GraphPaD Prism software was used and X was changed to logX to fit a dose-response curve, which is shown in
From the experimental results shown in
The c-Met protein was prepared with a diluent to a final concentration of 2 μg/ml, and added to a microplate at 100 μl per well, overnight at 4° C. The solution in the well was discarded, and the plate was washed three times with PBST. The plate was added with a blocking solution at 300 μl/well and incubated at 37° C. for 2 h. The solution in the well was discarded, and the plate was washed three times with PBST. AAJ8D6 antibody and ABT-700 (i.e., telisotuzumab, its sequence information can be found at https://extranet.who.int/soinn/mod/page/view.php?id=137&inn n=10366, the same below) were diluted to 9 concentrations, added to the 96-well plate and incubated for 2 h. The solution in the well was discarded, and the plate was washed three times with PBST. HRP detection antibody was prepared by adding diluent at a dilution of 1:5000, added at 100 μl per well, and incubated at 37° C. for 1 h. The solution in the well was discarded, and the plate was washed three times with PB ST. The plate was added with TMB color developing solution, incubated at room temperature for 2 min, and then added with Elisa stop solution. The plate was put on a reader to obtain the results, which are shown in Table 4 and
It can be seen from Table 4 and
The preparation of antibody-drug conjugates was performed according to a general conjugation method: a reducing agent and a protecting agent were prepared with purified water as follows: 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine) and 1-20 mM DTPA (Diethylene triamine pentacetate acid) mother liquor, where the amount of reducing agent can be added within a concentration range according to the required coupling rate, were mixed with a concentration of monoclonal antibody (such as: 5-30 mg/mL) according to a volume ratio (1:1), and the final concentration molar ratio of TCEP to antibody was 0.5-6.0:1. The reaction was performed by stirring at 25° C. for 1 h. The TCEP-reduced antibody can be directly conjugated.
A concentration (5 mM) of a linker-active drug unit compound was prepared and dissolved in 25% DMSO (dimethyl sulfoxide). According to the molar ratio of drug to thio group of 0.32.8:1, the drug was slowly added, and the reaction was performed by stirring at 25° C. for 1-4 h. After the reaction, centrifugal ultrafiltration was performed 3 times with PBS buffer to purify and remove residual unreacted drugs and free small molecules such as DMSO, and SDS-PAGE electrophoresis and hydrophobic high performance liquid chromatography (HIC-HPLC) were used to detect the conjugation.
The linker-active drug unit compounds used in this example are Mc-Val-Cit-PAB-MMAE, D07-Val-Cit-PAB-MMAE and Py-MAA-Val-Cit-PAB-MMAE, whose structural formula are respectively as follows (for the synthesis methods, reference can be made to patent applications CN108853514A (page 14 of the specification), CN111433188A (page 53 of the specification), and WO2019223579A1 (pages 25-27 of the specification)).
The following ADCs were prepared by the above method (p was an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8, q was an integer selected from 1, 2, 3 and 4, and Ab was the AAJ8D6 antibody provided by the present disclosure). The average DAR of these ADCs was 4.
In addition, this example also prepared an antibody-drug conjugate whose antibody part was ABT-700 antibody, the linking group was Mc-Val-Cit-PAB, and the active drug unit was MMAE, where p is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8, and the average DAR was 4.
In addition, this example also prepared an antibody-drug conjugate of human immunoglobulin IgG1, Mc-Val-Cit-PAB and MMAE (i.e., IgG1-Mc-Val-Cit-PAB), where p was an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8, and the average DAR was 4.
HT29 cells were resuspended in 6-well plates at approximately 1×105 cells per well. AAJ8D6 antibody, AAJ8D6-Mc-Val-Cit-MMAE, AAJ8D6-PY-Val-Cit-MMAE and AAJ8D6-D07-Val-Cit-MMAE were conjugated to pHAb amine-reactive dyes, respectively, and then diluted with cell culture medium to 10 μg/ml. 100 μl of AAJ8D6 antibody, AAJ8D6-Mc-Val-Cit-MMAE, AAJ8D6-PY-Val-Cit-MMAE and AAJ8D6-D07-Val-Cit-MMAE dye complexes were added to cells and incubated at 37° C. for indicated times (0 h, 1 h, 3 h, 5 h, 21 h and 24 h). The endocytosis effects of AAJ8D6 antibody, AAJ8D6-Mc-Val-Cit-MMAE, AAJ8D6-PY-Val-Cit-MMAE and AAJ8D6-D07-Val-Cit-MMAE were measured by flow cytometry. After 24 h, the samples were significantly endocytosed in HT29 cells; after 3 h, the endocytosis effects were all about 90%. It can be seen that the humanized antibody provided by the present disclosure and the prepared ADCs had good endocytosis effects, which will be able to efficiently deliver the drug into the target cells. The results are shown in
Cells MKN-45, SNU-620, HT29, and BXPC-3 with different c-Met expression levels in exponential doubling phase were seeded in 96-well plates, respectively, and incubated overnight in a 37° C., 5% CO2 incubator. AAJ8D6, AAJ8D6-Mc-Val-Cit-MMAE and IgG1-Mc-Val-Cit-PAB were prepared in complete medium to a concentration of 1.25-2000 ng/mL, and MMAE was prepared in complete medium to a concentration of 0.0021-80 ng/mL. They were added to the plate at 100 μL per well with triplicate wells set up for each concentration, and a blank control group was set as well. After 72±2 h of drug action, the plate was added with CCK-8 reagent, incubated at 37° C., 5% CO2 incubator for 1 to 4 h, and detected for OD value of each well at 450 nm of a microplate reader. The inhibition rate (IR) was calculated as: IR %=(OD of blank-OD of drug)×100/OD of blank. Four-parameter fitting was performed on the inhibition rate and drug concentration to calculate IC50.
Results and conclusions: As shown in
Example 7 In vitro efficacy differences between antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE and antibody-drug conjugate ABT-700-Mc-Val-Cit-MMAE
The suspension of human colon cancer cell line HT29 was added to a 96-well plate at a density of 100 μL/well and 5000 cells/well, and placed in a water-saturated 37° C., CO2 incubator overnight. The antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE and the antibody-drug conjugate ABT-700-Mc-Val-Cit-MMAE were serially diluted and added to a 96-well plate containing cells at 100 μL/well. The plate was placed in a 37° C. incubator to culture for another 72 h. The OD values at 450 nm were read with the microplate reader, and the inhibition rate (IR) was calculated as: IR %=(OD of blank-OD of drug)×100/OD of blank. The curve fitting software Softmax Pro7.0.3 Gxp was used to calculate IC50.
The results are shown in Table 5 and
42 Balb/c nude mice aged 6-7 weeks were taken, and 0.1 mL of MKN-45 cell suspension (2.4×106 cells/mouse) was subcutaneously implanted in the back of the right armpit of the mice. When the tumor grew to about 100-300 mm3, according to the tumor volume, mice were randomly divided into the Vehicle (PBS) group, IgG1-Mc-Val-Cit-PAB (3 mg/kg) group, antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE (0.75 mg/kg) group, antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE (1.5 mg/kg) group, antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE (3 mg/kg) and humanized antibody AAJ8D6 (3 mg/kg) group, 6 tumor-bearing mice in each group. Each group was administered intravenously, once a week, for a total of 3 times, and measured for long diameter and short diameter of tumor and body weight twice a week during the administration. On Day 21 after administration, the tumor inhibition rate (TGIRTV) was calculated for efficacy evaluation.
Calculation formula:
Tumor volume: TV=D1×D22/2, where D1 and D2 represent the long diameter and
the short diameter of the tumor, respectively;
Relative tumor volume: RTV=TVn/TV0, where TV° is the tumor volume before administration, and TVn is the tumor volume at each measurement;
Relative tumor inhibition rate: TGIRTV(%)=(1-TRTV/CRTV) 100%, TRTV means RTV of drug administration group or positive control group, and CRTV means RTV of negative control group.
Result and conclusion: The inhibitory effect of each experimental group and control group on MKN-45 subcutaneously transplanted tumor in nude mice is shown in Table 6 and
The mouse PDX (patient-derived xenograft) model of gastric cancer GA0046 was constructed by transplanting tumor tissue from human patients with gastric cancer into severely immunodeficient mice, in which the gastric cancer tissue grew to form a transplanted tumor.
The tumor-bearing mice with good tumor growth were picked and euthanized, and the tumors were removed and cut into small pieces (2-3 mm in diameter), which were then inoculated on the right shoulder of the mice to establish a subcutaneous gastric cancer model. The tumor growth was regularly observed, and when the tumor grew to an average volume of about 100-150 mm 3, according to the tumor size and the weight of mice, mice were randomly divided into Vehicle (PBS) group, antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE (10 mg/kg) group, and IgG1-Mc-Val-Cit-PAB (10 mg/kg) group, 6 tumor-bearing mice in each group. Each group was administered intravenously, once a week, for a total of 2 times, for a total of 21 times. The long diameter and short diameter of tumor and body weight were measured twice a week during the administration. On Day 21 after administration, the tumor inhibition rate (TGIRTV) was calculated for efficacy evaluation.
Calculation formula:
Tumor volume: TV=D1×D22/2, where D1 and D2 represent the long diameter and the short diameter of the tumor, respectively;
Relative tumor volume: RTV=TVn/TV0, where TV° is the tumor volume before administration, and TVn is the tumor volume at each measurement;
Relative tumor inhibition rate: TGIRTV(%)=(1-TRTV/CRTV) 100%, TRTV means RTV of drug administration group or positive control group, and CRTV means RTV of negative control group.
Results and conclusions: The tumor inhibitory effects of each experimental group and control group on the mouse gastric cancer PDX model (GA0046) are shown in Table 7 and
The mouse PDX (patient-derived xenograft) model of lung cancer LU2503 was constructed by transplanting tumor tissue from human patients with lung cancer into severely immunodeficient mice, in which the gastric cancer tissue grew to form a transplanted tumor.
The tumor-bearing mice with good tumor growth were picked and euthanized, and the tumors were removed and cut into small pieces (2-3 mm in diameter), which were then inoculated on the right shoulder of the mice to establish a subcutaneous lung cancer model. The tumor growth was regularly observed, and when the tumor grew to an average volume of about 150 mm3, according to the tumor size and the body weight of mice, mice were randomly divided into Vehicle (PBS) group, humanized antibody AAJ8D6 (10 mg/kg) group, and low, medium and high concentrations of antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE (1.1 mg/kg, 3.3 mg/kg, 10 mg/kg) groups, 6 tumor-bearing mice in each group. Mice in the antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE and humanized antibody AAJ8D6 groups were administered intravenously, once a week, for a total of 2 times. The long diameter and short diameter of tumor and body weight were measured twice a week during the administration. On Day 21 after administration, the tumor inhibition rate (TGIRTV) was calculated for efficacy evaluation.
Calculation formula:
Results and conclusions: The tumor inhibitory effects of each experimental group and control group on the mouse lung cancer PDX model (LU2503) are shown in Table 8 and
The above examples show that the antibody-drug conjugate AAJ8D6-Mc-Val-Cit-MMAE provided by the present disclosure has a very significant therapeutic effect on c-Met positive tumors.
The principles and implementations of the present disclosure are illustrated by specific examples herein, and the descriptions of the above examples are only used to help understand the method and the central idea of the present disclosure. Several improvements and modifications can also be made to the present disclosure without departing from the principles of the present disclosure, and these improvements and modifications also fall within the protection of the claims of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/112919 | Sep 2020 | WO | international |
| 202010918330.4 | Sep 2020 | CN | national |
The present application is the national phase of International Application No. PCT/CN2021/115490, titled “ANTI-C-MET ANTIBODY-DRUG CONJUGATE AND APPLICATIONS THEREOF”, filed on Aug. 31, 2021, which claims priority to PCT/CN2020/112919, filed on Sep. 1, 2020 with the World Intellectual Property Organization, which claims priority to Chinese Patent Application No. CN202010918330.4, filed on Sep. 2, 2020 with the China National Intellectual Property Administration, which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/115490 | 8/31/2021 | WO |
