WO2018086239A9 - 靶向tacstd2的抗体与药物偶联体(adc)分子 - Google Patents

靶向tacstd2的抗体与药物偶联体(adc)分子 Download PDF

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WO2018086239A9
WO2018086239A9 PCT/CN2017/000685 CN2017000685W WO2018086239A9 WO 2018086239 A9 WO2018086239 A9 WO 2018086239A9 CN 2017000685 W CN2017000685 W CN 2017000685W WO 2018086239 A9 WO2018086239 A9 WO 2018086239A9
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antibody
mmae
hrs7
drug
adc
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WO2018086239A1 (zh
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王春河
黄鹏
陈莉莉
贺梦影
陈艺丽
谭小蓉
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is in the field of biomedicine, and in particular, the present invention relates to antibodies and drug conjugate (ADC) molecules that target TACSTD2 and a general technique for optimizing the adapter.
  • ADC drug conjugate
  • ADCs Antibody-drug conjugates
  • ADCs are a class of biomacromolecules that use monoclonal antibodies to direct small molecule drug loading (drug or payload) to target cancerous or tumor tissue. Its general structural formula can be written as Ab-(L-D)n.
  • Ab is an antibody or other biomolecule with a targeting effect, such as a fusion protein, that specifically binds to a target on the surface of a tumor cell and carries the load into the cell.
  • a targeting effect such as a fusion protein
  • a target that is highly expressed on the surface of tumor cells should be selected to increase the number of ADC molecules that can enter the cell.
  • the expression of the target on normal cells should be as small as possible to reduce toxic side effects.
  • affinity, specificity, immunogenicity, and ability to induce antigen endocytosis of antibodies are also important.
  • D is a small molecule compound toxin, or a payload.
  • Many traditional tumor chemotherapy drugs have been used in the development of ADC molecules, but in the subsequent clinical trials, the expected anti-tumor activity is often not achieved, which may be due to the limited number of small molecules that ADC molecules can bring into tumor cells. Since the targeting of ADC can reduce the toxic side effects of small molecules, in recent years, the focus of ADC research and development has begun to turn to natural products and their derivatives with strong cytotoxicity. Because of their high toxicity, such molecules are often not clinically available [65].
  • microtubule synthesis inhibitors such as auristatins and maytansine, as well as calicheamicins, pyrrole and benzoxhenylenes.
  • PBD pyrrolobenzodiazepine
  • duocarmycin irinotecan analogs
  • SN-38 SN-38
  • nemoxine analogs such as PNU-159682.
  • the auristatin is a derivative of dolastoxin.
  • the rabbit toxin is a natural toxic polypeptide isolated from the marine shellless mollusk truncated sea rabbit. Its mechanism of killing cells is to inhibit cell aggregation by binding to tubulin, leading to cell cycle arrest and apoptosis. In addition to being rich in hydroxy acids, thiophenes, D-amino acids and ⁇ -amino acids, such polypeptide structures also contain olefinic and acetylenic bonds in some molecules. These groups increase the biological activity and stability of such compounds to some extent. Although the rabbit toxin compound has a large limitation when it is developed in a single drug form, its high cell killing ability just meets the requirements of the ADC molecule for the toxin molecule.
  • Monomethyl auristatin E is a synthetic pentapeptide derived from dolastatin-10 by converting its N-terminal tertiary amino structure to a secondary amino group which can be coupled to a linker and introducing a 2- at the C-terminus by synthesis. Amino-1-phenylpropyl-1-ol. MMAE maintains its original cell killing activity. In many human tumor cell lines, the IC50 is 10 -9 -10 -11 mol/L, which is 200 times that of the traditional small molecule chemotherapeutic drug vinblastine.
  • MMAE not only has strong growth inhibitory activity against a variety of human hematological malignancies and solid tumor cells (melanoma, lung cancer, gastric cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, colon cancer and kidney cancer). Moreover, in the above cell lines, the phenomenon of drug resistance is very small. After MMAE is coupled to the antibody via a cleavable linker, the selective inhibitory activity against positive tumor cells can still be 10-1000 times greater than that of the traditional chemotherapeutic drug doxorubicin. MMAE is now widely used in the development of ADC molecules.
  • MMAF auristatin toxoid monomethyl auristatin F
  • MMAF is also a synthetic derivative of dolastatin-10.
  • MMAF also contains a carboxyl group at the C-terminus that can be used as a coupling site.
  • carboxyl groups significantly enhances the ionization effect of MMAF and the metabolic rate of off-target toxins, which is of great significance for reducing the side effects of drugs.
  • MMAF has a significantly lower in vitro cell viability than MMAE due to poor membrane permeability; however, after coupling with antibodies, MMAF is brought into the cells by endocytosis of the antibody, which increases the killer cell activity.
  • the IC50 of monomeric MMAF was 68 nmol/L, while the cAC10-MMAF after antibody coupling was 0.021 nmol/L, an increase of 3200-fold: in the KMH-2 cell line.
  • the IC50 of the monomeric MMAF was 45.4 nmol/L, and the cAC10-MMAF coupled with the antibody was 0.009 nmol/L, and the activity was increased by 5000 times.
  • due to the poor penetrating ability of the molecule it is not easy to spread, so the penetration ability to cancer tissues is not as good as that of MMAE.
  • Maytansine is a natural product of benzoic acid, originally isolated from Ethiopian shrubs. These drugs also exert their cytotoxic effects by interfering with tumor cell microtubule assembly and inhibit tumor cell growth. In vitro, the cytotoxic activity of maytansinoid derivatives is 1000 times higher than that of clinically used anticancer drugs.
  • the structure modification of maytansine can be carried out by introducing a chemical group or the like which can be coupled to the antibody without affecting its cytotoxicity, and the resulting derivatives such as DM1 and DM4 can be used for coupling of the ADC.
  • Calicheamicin is an enediyne antibiotic isolated from Micromonospora echinospora SSP. Calichensis is one of the main components of Calicheamicin.
  • the biological activity of Calicheamicin includes low prophage-induced concentration ( ⁇ 1pmon/L), high antibacterial activity, and strong killing activity against mouse P3883, L1210 leukemia, Colon26 colon cancer, and B16 melanoma cell line.
  • the Calicheamicin molecule consists of two structural regions with biological functions. The larger structural region is an extended sugar-based structure comprising four monosaccharide units and a six-membered benzene ring.
  • the polarized iodine group on the six-membered substituted aromatic ring can cause Calicheamicin to produce its sequence-specific binding to the 5'TCCT 3' by interacting with two guanosine amino groups in the 3'AGGA 5' sequence of the DNA molecule.
  • Another structural region of the Calicheamicin molecule is its glycosidic moiety, which consists of a rigid enediyne core with two hydroxyl side chains called Calicheamicinone.
  • Calicheamicinone is a key component of the Calicheamicin molecule that produces biological activity, breaks DNA, and kills cancer cells.
  • the sulfhydryl group of the side chain of the Calicheamicin molecule can be chemically modified to be used for the coupling of ADC molecules.
  • PBD dimer Pyrrolobenzodiazepine
  • PBD dimer is a natural product extracted from soil actinomycetes. Its dimer can be inserted into the DNA ditch and form acetal with electrophilic C11 and guanine N3. The amine bond cross-links the two DNA strands together, thereby terminating the division of the tumor cells and ultimately leading to apoptosis.
  • PBD dimers are not selective for DNA sequences, and thus can overcome the drug resistance caused by tumor cell gene mutations. Recently, Seattle Genetics' Vadastuximab talirine (Anti-CD33-PBD dimmer) caused 6 cases (about 300 patients) of hepatotoxicity in phase III clinical trials, 4 of which died, making the safety of PBD dimers questionable.
  • Duocarmycin is a natural product with strong anticancer activity isolated from Streptomyces. They can be inserted into the small groove of DNA to alkylate the adenine in the DNA at the N3 position.
  • the vc-seco-DUBA from Synthon, the Netherlands, is a combination of a connector and a small molecule payload designed on the basis of the DNA inhibitor duocarmycin.
  • the ADC molecule SYD985 (Anti-HER2-vc-seco-DUBA) using this combination showed superior efficacy to T-DM1 both preclinically and clinically.
  • SN-38 is an active metabolite of the DNA topoisomerase I inhibitor irinotecan, which has anticancer activity and toxicity. Right lower. Immunomedics' sacituzumab govitecan (IMMU-132) chose SN-38 and achieved good clinical results.
  • Another DNA topoisomerase I inhibitor used in ADCs is PNU-159682, an active metabolite of the doxorubicin analog nemorubicin, but with a 3000-fold increase in activity compared to doxorubicin. NBE Therapeutics is a pioneer in the use of this load.
  • MDR multiple drug resistance protein
  • P-glycoprotein a multiple drug resistance protein
  • Multidrug resistance proteins are a plurality of glycoproteins expressed on the cell membrane, which can pump small molecule compounds such as MMAE and DM1 out of tumor cells, thereby making cells resistant.
  • Chemotherapy can up-regulate the expression of multidrug resistance protein in patients' tumor tissues, so small molecule drugs such as docamycin and PNU-159682, which are selected from non-multidrug resistance protein substrates, can partially overcome the upregulation of such proteins. The resulting resistance.
  • L is the linker, and the two functional modules of antibody and load are linked together by covalent coupling.
  • the cysteine or lysine residue of the antibody molecule itself can serve as a point of attachment.
  • the mode of coupling can be a non-site mode, ie the final product is a mixture of different numbers of load molecules coupled at different sites.
  • the two ADC molecules that are now available are coupled by a non-site method.
  • Site-directed coupling is the precise control of the loading of load molecules by adding artificial cysteine to the antibody molecule, modification of the sugar chain, incorporation of unnatural amino acids, or enzymatically modified modifications such as transpeptidase and transaminase modification. The number and location of the site.
  • the high efficiency release of the load in the ADC molecule is an important factor affecting the activity of the ADC molecule. This process is achieved by breaking the chemical link between the antibody and the load, so choosing the appropriate connector and coupling the appropriate number of loads at the appropriate site is the key to the ADC's efficacy.
  • the ideal connector is sufficiently stable in the peripheral blood circulation to avoid toxicity due to the release of cytotoxic molecules.
  • the ADC is endocytosed into the cell, it must be able to effectively release the load.
  • the connector of the ADC molecule can be classified into two types, cleavable and non-cleavable.
  • the cleavable connector in turn includes both chemical cleavage and enzymatic cleavage.
  • the commonly used chemical cleavage connector has an acid labile hydrazone bond head and a disulfide bond link: the dipeptide link is an enzyme catalytic cleavage link; the thioether bond and the amide bond are non-cleavable connectors.
  • Acid-labile hydrazone linkages are unstable under acidic conditions such as lysosomes and are more stable under neutral conditions in the peripheral circulation system. It is applied to the antibody-acanthomycin-conjugated ADC, and some of the sputum linkages have about 5%-6% of cytotoxic molecules shedding in the peripheral circulation system for 24 hours, and up to 97% in cancer cells. -98%. However, the half-life of the sputum linkage in the circulatory system is still relatively short (43 hours), and many naked antibodies themselves have a half-life in the human body for days or even weeks. Therefore, its selectivity is still relatively low. Disulfide bonds are also one of the commonly used connectors for ADCs. It is stable in the circulatory system and is cleaved under intracellular reducing conditions to release the load.
  • Dipeptide junctions such as valine-citrulline (vc) can be cleaved by specific lysosomal proteases, which are 100 times more stable than the chemically cleavable linker in the human circulatory system in mice.
  • the half-life in monkeys is about 6-10 days.
  • proline-citrulline exhibits relatively good stability in the blood, and is more susceptible to cleavage under the catalysis of cathepsin B in cell lysosomes, so it is currently the most widely used.
  • One of the ADC peptide chain linkers One of the ADC peptide chain linkers.
  • the thioether connector is a non-cleavable, stable connector. After the ADC using such a connector is internalized by tumor cells, the antibody portion is degraded in the lysosome, releasing a small molecule cytotoxic molecule with a thioether connector to exert an antitumor effect.
  • the use of thioether bonds can increase the stability and tolerability of ADC molecules in the blood circulation and intracellular environment, but the rate of release of small molecule toxins is slower and thiol exchange with human serum albumin, thereby limiting Its clinical application.
  • Tumor associated calcium signal transducer 2 (TACSTD2), also known as Trop-2, EGP-1, MS-1, Gp-15, T-16 and GA733-I, is a molecular weight of 45kDa A type I transmembrane glycoprotein in monomeric form. According to the gene sequence, its protein sequence contains a signal peptide (26 amino acid residues), an extracellular region (248 amino acid residues), a transmembrane region (23 amino acid residues) and an intracellular region (26 amino acid residues). base).
  • TACSTD2 In a variety of epithelial-derived solid tumors (such as breast cancer, cervical cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, and prostate cancer), TACSTD2 is overexpressed in a high proportion. For example, 76% of breast cancer, 55% of pancreatic cancer, and 56% of gastric cancer patients overexpress the antigen. Clinical data indicate that the expression level of TACSTD2 is positively correlated with tumor invasiveness and negatively correlated with patient prognosis. On cancerous cells, TACSTD2 is an oncogene with signaling function. It may be activated by antibody cross-linking or cleavage of intracellular fragment portions.
  • TACSTD2 Upon activation, its intracellular domain is phosphorylated by phosphokinase C and mediates the transmission of calcium signals.
  • Calcium signaling can activate or up-regulate a variety of signaling molecules that promote cell division and growth, such as NF ⁇ B, cyclin D-1, and ERKs, thereby accelerating cell growth, invasion, and metastasis.
  • NF ⁇ B NF ⁇ B
  • cyclin D-1 cyclin D-1
  • ERKs a variety of signaling molecules that promote cell division and growth
  • a large number of TACSTD2 are expressed in cancerous cells, and the expression varies greatly depending on the cell type, which may be a means of regulating TACSTD2 function. The large difference in the amount and function of TACSTD2 between tumor cells and normal cells makes it an ideal drug target.
  • ADC molecules Ab of most ADC molecules are hydrophilic, while small molecule toxin D generally uses hydrophobic compounds.
  • the difference in polarity between the two ends of the molecule is highly likely to cause aggregation of the ADC molecules, thereby affecting its drug-forming properties and pharmacodynamics.
  • the invention finds a specific method for improving the vc dipeptide connector by oligomeric PEG, which can balance the hydrophobicity of the small molecule toxin D, thereby improving the molecular medicinal properties and pharmacodynamics of the ADC, and can be used for various kinds. Coupling of different ADC molecules.
  • the object of the present invention is to provide (1) an antibody-to-drug conjugate (ADC) molecule targeting TACSTD2, which is a specific combination of antibodies hRS7 and MMAE, which combination has a significant synergistic effect; (2) provides a A dipeptide junction technology for optimized ADC molecules.
  • ADC antibody-to-drug conjugate
  • an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate structure being as shown in Formula I:
  • Ab represents an antibody and Ab is hRS7:
  • D is a cytotoxic small molecule drug selected from the group consisting of monomethyl auristatin, calicheamicin, maytansinoid, doxorubicin (doxorubicin) ), pyrrolobenzodiazepine (PBD), duocarmycin, or a combination thereof;
  • L is a linker that connects the antibody to the drug
  • n is the average number of couplings of the drug coupled to the antibody, may be an integer or a non-integer positive number, and 0.8 ⁇ n ⁇ 8;
  • n is the average number of coupled drugs coupled to the antibody, and is also the drug antibody ratio, also known as the DAR value.
  • n is an integer of 1-7 or a non-integer positive number.
  • n is an integer of 1-6 or a non-integer positive number: preferably, an integer of 2-6 or a positive number of a non-integer; more preferably, an integer or a number of 2-4 A positive number of integers.
  • D is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), doxorubicin, Pyrrole benzodiazepine, docamycin, or a combination thereof.
  • MMAE monomethyl auristatin-E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • doxorubicin Pyrrole benzodiazepine
  • docamycin or a combination thereof.
  • the Ab is a humanized antibody hRS7 that targets TACSTD2.
  • D is a cytotoxic small molecule drug monomethyl auristatin-E (MMAE).
  • MMAE cytotoxic small molecule drug monomethyl auristatin-E
  • L is a linker which can efficiently covalently couple hRS7 with MMAE.
  • n is a positive number of 1-6: preferably, a positive number of 2-6: more preferably, a positive number of 2-4.
  • D is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD) or monomethyl auristatin-F (MMAF).
  • MMAE monomethyl auristatin-E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • L is mc (maleimidocaproxyl, maleimidocaproyl)-vc (valine-citrulline, praline-citrulline)-PABC (para-aminobenzyloxycarbonyl).
  • L is MCC (maleimidomethyl cyclohexane-I-carboxylate, maleimide methylcyclohexyl-I-carboxylate group)-vc-PABC.
  • the vc connector is replaced by CL2A.
  • L is mc-vc-PABC
  • D is MMAE
  • the antibody-drug conjugate is hRS7-[mc-vc-PABC-MMAE]n (can be simply hRS7-[vc -MMAE]n or hRS7-vc-MMAE), where n is a positive number and its structure is as shown in Formula II:
  • L is comprised of x oligomeric polyethylene glycol PEG (represented by PEGx, where x is the number of repeats of polyethylene glycol PEG), and x is an integer greater than zero.
  • the antibody-drug conjugate when x oligo-glycol PEG is included in L, the antibody-drug conjugate can be represented as Ab-(L(PEGx)-D)n.
  • the PEGx may be selected from one or more of the group consisting of PEG2, PEG4, PEG6, PEG8, PEG12, PEG16, PEG24, PEG28, etc., wherein the number after PEG represents the number of repeats of PEG .
  • the antibody-drug conjugate is hRS7-[L(PEGx)-MMAE]n, wherein x is the number of repeats of PEG, preferably an integer from 1 to 28: preferably, An integer from 4 to 24.
  • the antibody-drug conjugate is hRS7-[mc-PEGx-vc-PABC-MMAE]n (abbreviated as hRS7-vc(PEGx)-MMAE), wherein x is a repeat of PEG The number, preferably an integer from 1 to 64; preferably, an integer from 4 to 24.
  • the PEG can be attached to maleic acid.
  • the antibody and drug conjugate are as shown in Formula III or IV:
  • more polyethylene glycol (PEGx) may be embedded in L to improve the hydrophilic properties of the molecule, such as Formula V-1, V-2, with a drug conjugate (ADC). As shown in V-3:
  • the citrulline (C) residue in L is replaced by a lysine, argine or glutamic acid residue, and then The oligo-PEG24 is attached to the reactive group of the amino acid side chain, and the molecule adopting the linkage mode shows the optimal drug-forming property and drug efficacy;
  • valine (V) residue in L when the valine (V) residue in L is subjected to an amino acid residue having a reactive group such as lysine, argine or Substituting a glutamic acid residue, then attaching PEGx to the reactive group of the amino acid side chain, as shown in Structural Formula VI, wherein x is an integer from 1 to 28 :n is a positive number from 1-8;
  • the antibody hRS7 has the following characteristics:
  • amino acid sequence of the light chain variable region of the antibody hRS7 comprises or is a sequence as set forth in SEQ ID NO.: 1;
  • amino acid sequence of the heavy chain variable region of the antibody hRS7 comprises or is the sequence set forth in SEQ ID NO.: 2.
  • a second aspect of the invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the invention, and a pharmaceutically acceptable carrier.
  • a third aspect of the invention provides the use of the antibody or drug conjugate of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention for the preparation of an antitumor drug.
  • the tumor includes, but is not limited to, breast cancer (Sanyin and non-three negative), gastric cancer, ovarian cancer, small cells, and non-small cells (squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma).
  • breast cancer Sanyin and non-three negative
  • gastric cancer gastric cancer
  • ovarian cancer small cells
  • non-small cells squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma.
  • a fourth aspect of the invention provides a method of preparing the antibody and drug conjugate of the first aspect of the invention, the method comprising the steps;
  • reaction system comprising an antibody and a drug molecule, the drug molecule being linked to a linker
  • the antibody and the drug molecule are subjected to a coupling reaction to prepare the antibody-drug conjugate.
  • the pH of the reaction system is from 6.0 to 8.0; preferably, from 6.0 to 7.0.
  • the molar ratio of the antibody to the drug molecule is from 1:1 to 1:20; preferably, from 1:10 to 1:20.
  • reaction time is from 1 h to 48 h; preferably, from 3 h to 36 h.
  • a fifth aspect of the invention provides a method of treating or preventing a tumor, the method comprising the steps of: administering to a subject in need thereof the antibody and drug conjugate of the first aspect of the invention or the second aspect of the invention medicine Composition.
  • the subject is a mammal, including a human.
  • the antibody and drug conjugate are administered at a concentration of from 0.3 to 10 mg/kg; preferably, from 1 to 6 mg/kg; more preferably from 1 to 4 mg/kg. .
  • a sixth aspect of the invention provides a universal PEGylated dipeptide linker technology.
  • a ligation L1 for linking small molecule drugs and biomacromolecules is provided, and L1 contains an ADC connection similar to vc (valine-citrulline, valine-citrulline) which can be cleaved by protease B.
  • vc valine-citrulline, valine-citrulline
  • L1 contains vc (valine-citrulline, valine-citrulline), wherein the citrulline residue is replaced by a lysine residue, and the active amino group of the lysine residue can be Connect x oligo-glycol PEG.
  • valine-citrulline can be cleaved by protease B.
  • L1 contains valine-lysine (VL).
  • VL valine-lysine
  • the reactive amino group of the lysine residue can be linked to x oligomeric polyethylene glycol PEG.
  • the x oligomeric polyethylene glycol PEGs can be represented by PEGx, wherein x is the number of repeats of the polyethylene glycol PEG and is an integer greater than zero.
  • L1 contains valine-lysine (m-dPEGx)-PAB (wherein PAB can also be represented as PABC). Wherein - is a bond or a linking group.
  • PEG is attached to the proline. Wherein PEG is linked to valine via a carbonyl group.
  • L1 is PEGx-valine-lysine (m-dPEGx)-PAB
  • L1 is PEG4-VL(m-dPEG24)-PAB.
  • ADC molecules using this modification have the best drug-forming and in vivo efficacy.
  • L1 further contains a maleimidohexanoyl group.
  • a maleimidocaproyl group is further attached to the proline.
  • the maleimidocaproyl group is attached to PEGx when PEGx is attached to the proline.
  • L1 is mc-PEG4-VL(m-dPEG24)-PAB.
  • the biomacromolecule has a targeting function
  • the biomacromolecule comprises an antibody, a fusion protein, and the like.
  • the small molecule drug is cytotoxic, and the small molecule drug includes, but is not limited to, monomethyl auristatin E-E, -D or -F (MMAE, MMAD or MMAF), calicheamicin, maytansinoids (DM1 and DM4, etc.), doxorubicin (doxorubicin), pyrrole benzodiazepine (PBD), docamycin (duocarmycin) , a nemorubicin analog (such as PNU-159682), or a combination of two or more small molecule drugs.
  • MMAE monomethyl auristatin E-E, -D or -F
  • calicheamicin calicheamicin
  • maytansinoids DM1 and DM4, etc.
  • doxorubicin doxorubicin
  • PPD docamycin
  • a nemorubicin analog such as PNU-159682
  • the structure of the linker connecting the small molecule drug and the biomacromolecule can be as shown in Formula I-1:
  • Ab is any biological macromolecule with a targeting function, including antibodies, fusion proteins, etc.:
  • D is a small molecule drug with cytotoxicity, including but not limited to: monomethyl auristatin E-E, -D or -F (MMAE, MMAD or MMAF), Gary , Maytansin (DM1 and DM4, etc.), doxorubicin (doxorubicin), pyrrole benzodiazepine (PBD), duocarmycin, nemorubicin Analogs (such as PNU-159682), or a combination of two or more small molecule drugs:
  • n is the average number of couplings of the drug coupled to the antibody, may be an integer or a non-integer positive number, and 0.8 ⁇ n ⁇ 8;
  • L1 is mc(maleimidocaproxyl, maleimidocaproyl)-vc(valine-citrulline, praline-citrulline-PABC (para-aminobenzyloxycarbonyl).
  • L1 is MCC (maleimidomethyl cyclohexane-I-carboxylate, maleimide methylcyclohexyl-I-carboxylate group)-vc-PABC.
  • the vc connector is replaced by CL2A.
  • the antibody-drug conjugate is hRS7-[mc-vc-PABC-MMAE]n (can be simply hRS7-[vc -MMAE]n or hRS7-vc-MMAE), where n is a positive number and its structure is as shown in Formula II:
  • x oligomeric polyethylene glycol PEG (represented by PEGx, where x is the number of repeats of polyethylene glycol PEG) is included in L1, and x is an integer greater than zero.
  • the antibody-drug conjugate when x oligo-glycol PEG is included in L1, the antibody-drug conjugate can be represented as Ab-(L1(PEGx)-D)n.
  • the PEGx may be selected from one or more of the group consisting of PEG2, PEG4, PEG6, PEG8, PEG12, PEG16, PEG24, PEG28, etc., wherein the number after PEG represents the number of repeats of PEG .
  • more polyethylene glycol (PEGx) may be embedded in L1 to improve the hydrophilic properties of the molecule, such as the formula V-1, V-2, and the drug conjugate (ADC). As shown in V-3:
  • the citrulline in L1 is replaced by a lysine, argine or glutamic-acid residue, and then the oligomeric PEGx is attached to On the reactive group of the amino acid side chain, the molecule adopting this linkage mode shows the optimal drug-forming property and drug effect.
  • the linker can be used for the coupling of a variety of different ADC molecules including, but not limited to, ADC molecules that target TACSTD2.
  • valine (V) residue in L1 when the valine (V) residue in L1 is subjected to an amino acid residue having a reactive group such as lysine, argine or Substituting a glutamic acid residue, then attaching PEGx to the reactive group of the amino acid side chain, as shown in Structural Formula VI, wherein x is an integer from 1 to 28 :n is a positive number from 1-8:
  • the connector L1 can be a connector or a linker in any of the ADC molecules given herein.
  • the connector L1 may be one or more of the connectors or connectors in the structural formula given in Embodiment 11.
  • Figure 1 shows that hRS7-vc-MMAE has significantly greater killing efficacy against pancreatic cancer cell BxPC-3 than hRS7-CL2A-SN38 in vitro.
  • MMAE itself is more damaging to tumor cells than SN38, it is in hRS7 After compatibility, this enhanced lethality was dramatically amplified, showing a significant synergy between the antibodies hRS7 and MMAE.
  • Figure 2 shows that hRS7-vc-MMAE showed a potent pharmacodynamic trend on a BALB/c-Nu tumor nude mouse model transplanted with 1x10 6 , 2x10 6 , 5x10 6 BxPC3.
  • Rx 3 mg/kg
  • Figure 3 shows that hRS7-vc-MMAE showed potent pharmacodynamics on a BALB/c-Nu tumor nude mouse model transplanted with MBA-MB-468.
  • QW, iv. Tumor volume length ⁇ width 2 /2.
  • n 5.
  • the 1 and 3 mg/kg dose groups were effective, while the 0.3 mg/kg dose group was not effective.
  • Figure 4 shows the killing efficiency of hRS7-vc-MMAE with different DAR values on breast cancer cell MBA-MB-468. Among them, the molecular effects of DAR values of 2, 4 and 6 are similar, and when the DAR value is lower than 2, the cell killing effect is not good.
  • Figure 5 shows that the ADC molecule hRS7-mc-PEG4-vc-PABC-MMAE (abbreviated as hRS7-vc(pEG4)-MMAE) with PEG4 embedded in the linker (or linker) has a more significant pharmacological effect.
  • Figure 6 shows the results of the stability experiments for each ADC molecule.
  • Figure 7 shows the experimental results of the stability of each ADC molecule.
  • Figure 8 shows the molecular hydrophobicity of each ADC molecule.
  • Figure 9 shows the molecular hydrophobicity of each ADC molecule.
  • Figure 10 shows the cell killing activity of each ADC molecule.
  • Figure 11 shows inhibition of mouse vaccination with MDA-MB-468 tumors by different PEG-modified Linker ADCs, wherein hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE has a good tumor suppressing effect.
  • Figure 12 shows that a PEG4-VL (m-dPEG24)-PAB linker can be used to couple different small molecule toxins.
  • the inventors have provided extensive screening through extensive and intensive research to obtain an efficient, loading, stability and hydrophobic optimized antibody and drug conjugate targeting TACSTD2.
  • the experimental results show that the antibody and drug conjugate of the present invention achieve an abnormally significant antitumor effect by rationally matching the antibody hRS7 with a small molecule drug (such as MMAE); on the other hand, using an optimized loading amount, significant The toxic side effects of the antibody of the present invention and the drug conjugate against other cells such as normal cells are reduced.
  • the polyethylene glycol is embedded in the citrulline position in the dipeptide junction of the conjugate, which further improves the physicochemical properties of the molecule and improves the drug effect.
  • the linker technology can be used for the coupling of various ADC molecules. The present invention has been completed on this basis.
  • antibody to drug conjugate of the invention As used herein, the terms “antibody to drug conjugate of the invention”, “antibody of the invention and drug conjugate”, “conjugate of the invention” or “ADC of the invention” are used interchangeably and mean An antibody-drug conjugate as shown in the structure of Formula I. Further, in the present invention, the same or the same type of ADC molecule can be represented in various forms.
  • one of the ADC molecules, hRS7-[mc-vc-PABC-MMAE]n when n is a different value, can be generically referred to as hRS7-vc-PAB-MMAE or hRS7-mc-vc-PABC-MMAE or hRS7 -vc-MMAE or hRS7-mal-vc-PABC-MMAE.
  • the value of n in hRS7-[mc-vc-PABC-MMAE]n is the DAR value of the ADC molecule.
  • DAR is 2 hRS7-mc-vc-PABC-MMAE or hRS7-vc-MMAE, ie hRS7-[mc-vc-PABC-MMAE] 2 .
  • hRS7-vc(PEG4)-MMAE can be represented as hRS7-mc-PEG4-vc-PABC-MMAE or hRS7-PEG4-vc-PAB-MMAE.
  • Mal-Peg4-Val-Lys(m-dPEG24)-PAB-MMAE can also be expressed as Mal-Peg4-Val-Lys(PEG24-Me)-PABC-MMAE
  • the ADC molecule formed especially with hRS7 can be expressed as hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE;
  • Mal-Peg4-Lys(Peg24-Me)-Cit-PAB-MMAE can also be expressed as Mal-Peg4-Lys(PEG24-Me)-Cit-PABC-MMAE, especially the ADC molecule formed with hRS7 can be expressed as hRS7-PEG4 -L(m-dPEG24)-C-PAB-MMAE;
  • Mal-PEG24-VC-PAB-MMAE can also be expressed as Mal-PEG24-Val-Cit-PABC-MMAE, and the ADC molecule formed especially with hRS7 can be expressed as hRS7-PEG24-vc-PAB-MMAE.
  • PEGx can also be expressed as dPEGx.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. 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 a plurality of constant regions.
  • VH variable region
  • 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 to the variable region of the heavy chain. .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • the antibody portion of the ADC molecule can be replaced by a targeting fusion protein.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Different types of immunoglobulins The bit structure and three-dimensional configuration are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the antibody (or a fragment thereof) of the present invention which is encoded by the chemical synthesis.
  • the DNA sequence of or a derivative thereof can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem., 107: 220 (1980).
  • the antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibody that targets TACSTD2 is the antibody hRS7.
  • the light chain variable region (V-Kappa) amino acid sequence of the antibody hRS7 is the amino acid sequence set forth in SEQ ID NO.: 1 (DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ).
  • the heavy chain variable region (VH) amino acid sequence of the antibody hRS7 is the amino acid sequence (SEQ. s.
  • Small molecule drugs suitable for use in the present invention are compounds having high cytotoxicity, preferably monomethylaustatatin, calicheamicin, maytansin, doxorubicin (doxorubicin), pyrrole benzene Pyrrolobenzodiazepine (PBD), duocarmycin, or a combination thereof: more preferably: monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF) or a combination thereof.
  • MMAE monomethyl auristatin-E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • a linker (L or L1) suitable for use in the present invention is used to link the small molecule drugs and antibodies of the present invention.
  • L or L1 is mc-vc-PABC (abbreviated as vc).
  • L or L1 is MCC-vc-PABC.
  • the x or L1 of the present invention may contain x oligomeric polyethylene glycols (which may be represented by PEGx, where x is the number of repeats of polyethylene glycol (PEG)). Where x can be an integer from 1-28.
  • the PEGx may be selected from one or more of the group consisting of PEG2, PEG4, PEG6, PEG8, PEG12, PEG16, PEG24, PEG28, etc., wherein the number after PEG represents the number of repeats of PEG .
  • the PEG can be attached to maleic acid.
  • the present invention provides an antibody-to-drug conjugate (ADC) comprising (a) an antibody that targets TACSTD2 and (b) a cytotoxic small molecule drug linked together by a linker (L or L1).
  • ADC antibody-to-drug conjugate
  • the present invention provides a coupling method for coupling a small molecule drug to an antibody through a specific linker, and greatly improving the lethality of the antibody against tumor cells without changing the affinity of the antibody.
  • Typical coupling methods suitable for use in the present invention include both K-Lock and C-Lock coupling methods.
  • K lysine
  • C cysteine in the antibody sequence
  • the invention employs a specific linker (L or L1) moiety that is capable of coupling to a particular lysine to obtain a site-coupled, load-optimized antibody coupling. drug.
  • the preparation method of the antibody and drug conjugate of the present invention is as follows:
  • Aspirate a certain amount (such as 0.5 mg) of the antibody add 2 times the equivalent of Tris (2-carboxyethyl) phosphine (TCEP), and react at 37 ° C for a period of time (such as 2.5 hours) .
  • the reaction mixture was concentrated to less than 0.5 ml by centrifugal ultrafiltration, and filled up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • We finally replaced the buffer with a pH 7.0 PBS solution via an Amicon microliter Tra 4 10K ultrafiltration tube.
  • the preparation method of the antibody and drug conjugate of the present invention is as follows:
  • TCEP 2,-carboxyethyl) phosphine
  • the preparation method of the antibody and drug conjugate of the present invention is as follows:
  • a certain amount (such as 0.5 mg) of the antibody is aspirated, and 3 times the substance equivalent of dithiothreitol (DTT) is added, and the reaction is carried out at 37 ° C for a certain period of time (for example, 2 hours).
  • the reaction mixture was concentrated to less than 0.5 ml by centrifugal ultrafiltration, and filled up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • a coupling solution 75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO
  • We finally replaced the buffer with a pH 7.0 solution in PBS via an Amicon Ultra 4 10K ultrafiltration tube.
  • L or L1 is mc-vc-PABC, and when D is MMAE, the antibody and the drug are The conjugate is hRS7-[mc-vc-PABC-MMAE]n (abbreviated as hRS7-[vc-MMAE]n or hRS7-vc-MMAE), wherein n is a positive number of 1-8, and its structure is as follows Formula II;
  • the antibody-drug conjugate when x oligo-glycol PEG is included in L or L1, the antibody-drug conjugate can be represented as Ab-(L(or L1)(PEGx)-D) n.
  • the antibody-drug conjugate is hRS7-[L(or L1)(PEGx)-MMAE]n, wherein x is the number of repeats of PEG, preferably an integer from 1-28; Good place, an integer of 4-24.
  • the antibody-drug conjugate is hRS7-[mc-PEGx-vc-PABC-MMAE]n (abbreviated as hRS7-vc(PEGx)-MMAE), wherein x is a repeat of PEG The number, preferably an integer from 1 to 64; preferably, an integer from 4 to 24.
  • the antibody and drug conjugate are as shown in Formula III or IV:
  • more poly polyethylene glycol (PEG) may be embedded in L (or L1) to improve the hydrophilic properties of the molecule, such as the formula V-1. , V-2, V-3:
  • the invention also provides pharmaceutical compositions comprising the ADCs of the invention, and methods of using the ADCs of the invention to treat diseases in a mammal.
  • the disease is a disease targeted by TACSTD2, such as a plurality of malignant solid tumors.
  • the invention also provides the use of the antibody and the drug conjugate in the preparation of an antitumor drug.
  • the pharmaceutical composition comprises an effective amount of an ADC (as an active ingredient) according to the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.
  • the active ingredient is usually mixed with excipients, or diluted with excipients, or enclosed in a carrier in the form of a capsule or sachet.
  • the excipient acts as a diluent, it can be employed as a vehicle for the excipient, carrier or active ingredient in solid, semi-solid or liquid materials.
  • the composition may be a solution, a sterile injectable solution or the like.
  • Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; the formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl hydroxybenzoate and propyl ester) and the like.
  • the anti-tumor drug can be formulated in a unitary or multi-dose form, each dosage form comprising a predetermined amount of an ADC of the invention calculated to produce the desired therapeutic effect, as well as a suitable pharmaceutical excipient.
  • the antitumor drug can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration and the like.
  • a safe and effective amount of the antibody and drug conjugate is administered to a human, wherein the safe and effective amount is preferably in the range of 0.5 to 50 mg/kg body weight, more preferably 1 to 10 mg/kg body weight.
  • the specific dose should also consider the route of administration, the health of the patient and other factors, which are within the skill of the skilled physician.
  • conjugates of the present invention may also be combined with other therapeutic agents, including but not limited to: various cytokines such as TNF, IFN, IL-2, etc.; various tumor chemotherapy drugs, such as 5- FU, methotrexate and other drugs that affect nucleic acid biosynthesis; alkylating agents such as nitrogen mustard and cyclophosphamide; drugs such as doxorubicin and actinomycin D that interfere with transcriptional processes to prevent RNA synthesis: vincristine, camptothecin Classes and other drugs that affect protein synthesis and certain hormone drugs, and so on.
  • various cytokines such as TNF, IFN, IL-2, etc.
  • various tumor chemotherapy drugs such as 5- FU, methotrexate and other drugs that affect nucleic acid biosynthesis
  • alkylating agents such as nitrogen mustard and cyclophosphamide
  • drugs such as doxorubicin and actinomycin D that interfere with transcriptional processes to prevent RNA synthesis: vincristine, camptothecin Classes
  • the antibody and the drug conjugate provided by the present invention have a very prominent activity for killing tumor cells on tumor cells, and the synergistic effect of the antibody and the small molecule toxin is remarkable.
  • the antibody and drug conjugates provided by the present invention have optimized DAR values and better loading, making the therapeutic window of the ADC of the present invention larger.
  • the antibody and drug conjugates provided by the present invention have an optimized DAR value of 2-4, which retains more disulfide bonds in the antibody than the high DAR value ADC, so that the ADC of the present invention has more High stability.
  • the present invention introduces polyglycol in the citrulline moiety of the linker, thereby remarkably improving the physicochemical properties of the molecule and improving the drug effect.
  • the antibody and drug conjugate provided by the present invention have better pharmacokinetic properties and lower toxic side effects.
  • Isotype refers to an isotype control antibody, here a humanized antibody that targets ToxinB, belonging to the human IgGI antibody type.
  • MDA-MB-468 cells are conventional triple-negative breast cancer cells purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • BxPC-3 is a conventional pancreatic cancer cell and can be purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • HEK293F cells were purchased from Thermo Fisher China Ltd.
  • CHO-K1 cells were purchased from ATCC, USA.
  • mc-vc-PABC-MMAE was purchased from Nanjing Lianning Biomedical Technology Co., Ltd.
  • hRS7-CL2A-SN38 For the preparation method, see US Pat. No. 8,758,752 B2.
  • the light chain (amino acid sequence shown in SEQ ID NO: 1) and heavy chain (amino acid sequence shown in SEQ ID NO.: 2) of the hRS7 antibody were first cloned into the expression vector pcDNA3.1. Then, the plasmids carrying the cDNA sequences of the antibody light and heavy chains, respectively, were amplified in E. coli, and then HEK293 or CHO cells suspended in serum-free medium were transfected with the transfection reagent PEI, and at 37 ° C, Incubate for 4-10 days under 5% CO 2 conditions.
  • the antibodies in the cell culture supernatant were separated and purified on a GE AKTA Purifiier using Protein A and Superdex 200 size exclusion chromatography columns, and finally concentrated by ultrafiltration to obtain an antibody protein solution at a concentration of 1 mg/ml or more.
  • the ADC molecule hRS7-vc-MMAE with different DAR values can be prepared by changing the conditions of reduction and coupling as follows.
  • vc-MMAE is mc-vc-PABC-MMAE.
  • the antibody was concentrated to 5-10 mg/ml in PBS (pH 7.0) buffer by ultrafiltration. According to the measured concentration, 1 mg of the antibody was taken up, and 1.5 times the equivalent of tris(2-carboxyethyl)phosphine (TCEP) was added to react at 37 ° C for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to less than 0.5 ml by centrifugal ultrafiltration, and filled up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • TCEP tris(2-carboxyethyl)phosphine
  • the ADC molecule hRS7-vc-MMAE (1) prepared by this method has a DAR value of about 2.
  • the antibody was concentrated to 5-10 mg/ml in PBS (pH 7.0) buffer by ultrafiltration. According to the measured concentration, 1 mg of the antibody was taken up, and 10 times the equivalent of tris(2-carboxyethyl)phosphine (TCEP) was added to react at 37 ° C for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to less than 0.5 ml by centrifugal ultrafiltration, and filled up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • TCEP tris(2-carboxyethyl)phosphine
  • the ADC molecule hRS7-vc-MMAE (2) prepared by this method has a DAR value of about 4.
  • the antibody was concentrated to 5-10 mg/ml in PBS (pH 7.0) buffer by ultrafiltration. According to the measured concentration, 1 mg of the antibody was taken up, and 25-fold equivalent of tris(2-carboxyethyl)phosphine (TCEP) was added to react at 37 ° C for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated to less than 0.5 ml by centrifugal ultrafiltration, and filled up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • TCEP tris(2-carboxyethyl)phosphine
  • the antibody was concentrated to 5-10 mg/ml in PBS (pH 7.0) buffer by ultrafiltration. According to the measured concentration, 1 mg of the antibody was taken up, and 0.5-fold equivalent of tris(2-carboxyethyl)phosphine (TCEP) was added to carry out a reaction at 37 ° C for 2.5 hours. After the completion of the reaction, we concentrated the reaction mixture to below 0.5 ml by centrifugal ultrafiltration, and filled it up with a coupling solution (75 mM NaAc, pH 6.5, 1 mM DTPA, 10% DMSO), and then concentrated by centrifugation to 0.5 ml or less. three times.
  • TCEP tris(2-carboxyethyl)phosphine
  • Each of the ADC molecules hRS7-vc-MMAE (5) was prepared by the above method, and the DAR value was about 2.8, except that TCEP reduction of 6 times the substance equivalent of the antibody was added, and 10 times the material equivalent of vc-MMAE was added.
  • Pancreatic cancer cell BxPC3 is 10,000 cells/well
  • breast cancer MDA- MB-468 is 20,000/hole
  • the drug molecules were diluted 1:4 in the medium at different initial concentrations. Each of the three wells was administered at a concentration (200 ⁇ l total volume).
  • Control group hRS7-CL2A-SN38 (DAR value is about 2.8)
  • pancreatic cancer cell BxPC3 The killing efficacy of pancreatic cancer cell BxPC3 was compared in the drug-administered group and each control group in vitro (results shown in Figure 1).
  • MMAE is more powerful than SN38, and the EC 50 values of the two are 0.27 and 6.9 nM, respectively, which differ by more than 20 times.
  • the pharmacodynamic difference between MMAE and SN38 was dramatically amplified when constructed in an ADC molecule with hRS7 as an antibody.
  • EC 50 values hRS7-vc-MMAE anti BxPC3 cells is 0.27nM, and hRS7-CL2A-SN38 is greater than 300nM.
  • the tumor cell killing efficacy of hRS7-vc-MMAE was increased by 1111 times (Table 1).
  • Pancreatic cancer cell line BxPC-3 or breast cancer cell MDA-MB-468 was diluted with physiological saline to the required cell concentration, and subcutaneously injected into the axillary site of female BALB/c-nu nude mice aged 4-6 weeks. 6 / 300 microliters of tumor cells. Tumor size was measured daily using a vernier caliper after inoculation.
  • Tumor volume Lx ⁇ 2 /2
  • L is the longest diameter and ⁇ is the shortest diameter.
  • Treatment trials were initiated 36 days after inoculation or when the tumor volume approached 0.25 cm3 .
  • ADC molecules are typically injected once a week (QW), 0.3-3 mg/kg body weight.
  • QW time to which the tumor is larger than 1.0 cm 3 , the mouse is considered dead.
  • the therapeutic effect can be divided into partial reactions (tumor shrinkage of 30% or more), stable (tumor shrinkage of 29% or less, or growth of no more than 20%).
  • TTP tumor growth time
  • Statistical analysis of tumor growth is based on area under the curve (AUC). The statistical method was a two-tailed Student's-test to compare each of the administration groups with the control group.
  • ADC hRS7-vc-MMAE, DAR values were approximately 2.8 and 4 in pancreatic cancer and breast cancer, respectively
  • Control group isotype-ADC (isotype-vc-MMAE, DAR values were approximately 2.8 and 4 in pancreatic and breast cancer, respectively)
  • hRS7-CL2A-SN38 (DAR values are approximately 2.8 and 4 in pancreatic and breast cancer, respectively)
  • Example 1 The in vivo antitumor activity of the ADC molecule prepared in Example 1 was tested separately.
  • the experimental method was the same as that of Example 4, except that the tumor cells used in this experiment were breast cancer cells MBA-MB-468.
  • the results show that the ADC molecules prepared by the invention have excellent tumor killing efficacy at 4 or more.
  • Example 5 The lethality of hRS7-vc-MMAE molecule embedded in PEG4 on MBA-MB-468 cells
  • the antibody was coupled to the small molecule Mal-PEG4-vc-PABC-MMAE to form hRS7-vc(PEG4)-MMAE with different DAR values.
  • the coupling method is shown in Example 1.
  • Fmoc-Val-Lys(Trt)-PAB-PNP 100 mg, 0.102 mmol, purchased from Levena Biopharma, USA
  • MMAE 78 mg, 0.102 mmol, purchased from Levena Biopharma, USA
  • DIEA 7 mg was added to the solution.
  • the mixture was reacted at room temperature (22 ° C) for 2 hours, and the objective product Fmoc-Val-Lys(Trt)-PAB-MMAE( 3 ) was directly purified by reverse-HPLC.
  • the product ( 3 ) formed as a white powder (123 mg, 77%) after lyophil.
  • Fmoc-val-Lys-PAB-MMAE TFA salt (ie compound 4) (80 mg), m-dPEG24 acid (69 mg) and HATU (23 mg) were dissolved in DMF (3 ml) and added to DIEA (20 mg) at room temperature After stirring for 30 minutes, the desired product [MS: m/z 1208.8 (M / 2+H + )] was obtained. Diisopropylamine (1 mL) was added to the reaction mixture and stirred at room temperature for 3 hr. The mixture was reacted, concentrated and purified by reversed-purpur, and lyophilized compound 5 (53 mg). MS: m/z 1097.3 (M / 2+H + ).
  • the antibody was conjugated to compound 6 to the ADC molecule hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE by the method of Example 1.
  • H-Cit-PAB-MMAE (ie compound 2') (40 mg), Fmoc-Lys(Boc)-OH (18 mg, purchased from Chem-Impex Inc., USA) and HATU (15 mg) were dissolved in DMF (3 mL) DIEA (15 mg) was added and stirred at room temperature for 20 minutes. The crude product was purified by HPLC to give the desired product Fmoc-Lys(Boc)-Cit-PAB-MMAE (41 mg). MS: m/z 1474.3 (M + H + ).
  • Example 2 Following the procedure of Example 1, the antibody was conjugated to compound 5' to the ADC molecule hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE.
  • Example 2 Following the procedure of Example 1, the antibody was coupled to compound iii to the ADC molecule hR S7-PEG24-vc-PAB-MMAE.
  • the mobile phase of UHPLC was 50 mM phosphate buffer (pH 7.0) + 150 mM NaCl, column temperature 25 ° C, injection volume 20 ⁇ L, flow rate 1 ml/min, UV detection wavelengths 214 nm and 280 nm.
  • the stability of the ADC molecule was lowered by the coupling of small molecules, and more polymerized and melted molecules appeared than the antibody hRS7 (A and B).
  • the present invention adds a plurality of PEGs to the linker to reduce the hydrophobicity of the molecule.
  • PEG can be added to the linker in a variety of ways. I We used a hydrophobic column, hydrophobic interaction chromatography (HIC), which validated this hypothesis on HPLC and optimized the way PEG was inserted.
  • HIC hydrophobic interaction chromatography
  • the data in Figure 9 shows that PEGylation can alter the hydrophobicity of the ADC molecule.
  • the hydrophobicity of hRS7-PEG4-vc-PAB-MMAE, hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE and hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE relative to hRS7-vc -PAB-MMAE has been reduced.
  • hRS7-PEG24-vc-PAB-MMAE has a poor effect on the improvement of molecular hydrophilicity.
  • a decrease in hydrophobicity can reduce the tendency of ADC molecules to aggregate in solution by hydrophobic small molecule interactions.
  • the decrease in hydrophobicity can reduce the rate at which the ADC molecules are excreted by the liver, thereby prolonging the half-life of the molecule in the body, enhancing the efficacy, and reducing the toxic side effects on the liver.
  • the breast cancer cell line MDA-MB-468 cells was diluted with physiological saline to the desired concentration, subcutaneously in female BALB 4-6 weeks of age c-nu nude mice injected axillas / 1-5x10 6 cells / 300 microliters Tumor cells. Tumor size was measured daily using a vernier caliper after inoculation.
  • Tumor volume Lx ⁇ 2 /2
  • L is the longest diameter and ⁇ is the shortest diameter.
  • Treatment trials were initiated 36 days after inoculation or when the tumor volume approached 0.25 cm3 .
  • ADC molecules are typically injected once a week (QW), 0.3-3 mg/kg body weight.
  • QW time to which the tumor is larger than 1.0 cm 3 , the mouse is considered dead.
  • the therapeutic effect can be divided into partial reactions (tumor shrinkage of 30% or more), stable (tumor shrinkage of 29% or less, or growth of no more than 20%).
  • TTP tumor growth time
  • Statistical analysis of tumor growth is based on area under the curve (AUC). The statistical method was a two-tailed Student's-test to compare each of the administration groups with the control group.

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Abstract

靶向TACSTD2的抗体与药物偶联体(antibody drug conjugate，ADC)分子以及一种通用的成药性好的ADC连接头(linker)技术。具体地，公开了一种优化的靶向TACSTD2的ADC分子、其制备方法、制药用途和在抑制或治疗肿瘤中的作用。实验结果表明，所述ADC分子具有优于同类分子的抗肿瘤效果。同时，还公开了一种优化的、对含有vc(valine-citrulline，缬氨酸-瓜氨酸)的ADC连接头进行特定PEG修饰的方法，以提高ADC分子的可开发性和药效。

Description

靶向TACSTD2的抗体与药物偶联体(ADC)分子 技术领域
本发明属于生物医药领域，具体地说，本发明涉及靶向TACSTD2的抗体与药物偶联体(ADC)分子以及一种通用的对连接头优化的技术。
背景技术
抗体与药物偶联体(antibody-drug conjugate，ADC)是应用单克隆抗体来引导小分子化合物载荷(drug或payload)靶向性地杀伤癌变细胞或肿瘤组织的一类生物大分子药物。其通用结构式可以写为Ab-(L-D)n。
Ab为抗体或其它具有靶向作用的生物大分子如融合蛋白，可以特异性地结合肿瘤细胞表面的靶点并把载荷携带到细胞内。为了达到理想的治疗效果，应该选择在肿瘤细胞表面高表达的靶点，以增加可以进入细胞内的ADC分子数目。同时，靶点在正常细胞上的表达量应该尽可能少，以减轻毒副作用。另外，抗体的亲和力、特异性、免疫原性和诱导抗原内吞的能力也很重要。
D为小分子化合物毒素，或称做载荷(payload)。许多传统的肿瘤化疗用药已经被用于ADC分子的研发，但是在其后的临床试验中往往无法达到预期的抗肿瘤活性，这可能是因为ADC分子可以带入肿瘤细胞内的小分子有限。由于ADC的靶向性可以降低小分子的毒副作用，因此，近年来ADC的研发重点开始转向具有强力细胞毒性的天然产物及其衍生物。这类分子因为毒性过高，往往在临床上无法单独使用[65]。在ADC分子中应用较多的载荷分子有奥利他汀类(auristatins)和美登素类(maytansine)等微管合成抑制剂，以及刺孢霉素类(calicheamicins)、吡咯开苯并吖庚三烯(pyrrolobenzodiazepine，PBD)、多卡霉素(duocarmycin)、伊立替康类似物如SN-38、以及奈莫柔比星类似物如PNU-159682等DNA抑制剂。
奥利他汀类化合物是海兔毒素(dolastoxin)的衍生物。海兔毒素是从海洋无壳软体动物截尾海兔中分离提取的天然毒性多肽，它杀伤细胞的机制是通过与微管蛋白结合，抑制其聚合，从而导致细胞周期阻滞及细胞凋亡。此类多肽结构除了包含丰富的羟基酸、噻酚、D型氨基酸与α-氨基酸之外，部分分子中还含有烯键与炔键。这些基团在一定程度上提高了此类化合物的生物活性及稳定性。虽然海兔毒素类化合物在以单药形式开发时存在较大的局限性，但其高效的细胞杀伤能力恰好满足了ADC分子对毒素分子的要求。
Monomethyl auristatin E(MMAE)是由海兔毒素-10衍生的合成五肽，通过将其N端的叔氨基结构转换为可与连接头相偶联的仲氨基，并通过合成在C端引入一个2-氨基-1-苯基丙基-1-醇。MMAE较好地保持了原有的细胞杀伤活力，在多种人肿瘤细胞系中，IC50为10-9-10-11mol/L，是传统小分子化疗药物长春碱的200倍。MMAE不仅对多种人血液恶性肿瘤和实体瘤细胞(黑素瘤、肺癌、胃癌、前列腺癌、卵巢癌、胰腺癌、乳腺癌、结肠癌和肾癌等)均具有较强的生长抑制活性，并且在上述细胞系中，产生耐药的现象非常少。MMAE通过可裂解的连接头与抗体偶联之后，对阳性肿瘤细胞选择性抑制活性仍可超过传统化疗药物多柔比星的10-1000倍。MMAE现已被广泛应用于ADC分子的开发之中。
另一个常用的auristatin类毒素monomethyl auristatin F(MMAF)也是海兔毒素-10的合成衍生物。除了与MMAE一样包含仲氨基偶联位点之外，MMAF在C端还包含一个极性较大，同时可以作为偶联位点的羧基。羧基的引入较为明显地提升了MMAF的离子化效应和脱靶毒素的代谢速率，这对减小药物的毒副作用有着非常重要的意义。研究表明，单体的MMAF由于透膜能力较差，体外细胞活性较MMAE显著降低；但与抗体偶联之后，通过抗体的内吞作用将MMAF带入细胞，使其杀伤细胞活性提高。如在CD30表达阳性的细胞系L428中，单体MMAF的IC50为68nmol/L，而与抗体偶联后的cAC10-MMAF则为0.021nmol/L，提高了3200倍：在KMH-2细胞系中，单体MMAF的IC50为45.4nmol/L，与抗体偶联后的cAC10-MMAF为0.009nmol/L，活性更是提高了5000倍。然而，由于该分子的穿膜能力差，不易扩散，因此对癌组织的穿透能力不如MMAE。
美登素(maytansine)属于苯酸类天然产物，最早分离自埃塞俄比亚灌木。这类药物也是通过干扰肿瘤细胞微管装配发挥其细胞毒性效应，抑制肿瘤细胞生长。在体外实验中，美登素类衍生物的细胞毒活性比临床常用抗癌药物高1000倍。在不影响其细胞毒性的前提下，通过引入可与抗体偶联的化学基团等，对美登素进行结构修饰，产生的衍生物如DM1和DM4可用于ADC的偶联。
Calicheamicin属于烯二炔类抗生素，分离自Micromonospora echinospora SSP。Calichensis是Calicheamicin的主要组分之一。Calicheamicin的生物活性包括低原噬菌体诱导浓度(＜1pmon/L)、高抗菌活性、对小鼠P3883、L1210白血病，Colon26结肠癌，B16黑色素瘤细胞株等均具有很强的杀伤活性。Calicheamicin分子由两个具有生物学功能的结构区域组成。较大的结构区域是一个伸展的糖基结构，包括四个单糖单位和一个六元取代苯环。其中，六元取代芳香环上的极化碘基团可以通过与DNA分子3′AGGA 5′序列中两个鸟嘌呤氨基相互作用，使Calicheamicin产生其与5′TCCT 3′的序列特异性结合。Calicheamicin分子的另一个结构区域是其糖苷元部分，由一个带有两个羟基侧链的刚性烯二炔核心构成，称为Calicheamicinone。Calicheamicinone是Calicheamicin分子产生生物学活性、断裂DNA、杀伤癌细胞的关键性组分。Calicheamicin分子侧链的巯基基团经化学修饰后可用于ADC分子的偶联。
吡咯苯并二氮杂卓(pyrrolobenzodiazepine，PBD)是一类从土壤放线菌中提取的天然产物，其二聚体可以嵌入DNA小沟中，通过亲电子的C11与鸟嘌呤的N3形成缩醛胺键，将两条DNA链共价交联在一起，从而使肿瘤细胞的分裂终止，最终导致细胞凋亡。PBD二聚体对DNA序列没有选择性，因此可以克服肿瘤细胞基因突变带来的耐药性。最近，Seattle Genetics公司的Vadastuximab talirine(Anti-CD33-PBD dimmer)在III期临床中导致6例(300左右总病人)肝毒性，其中4人死亡，使PBD二聚体的用药安全性受到质疑。
多卡霉素(duocarmycin)是从链霉菌中分离到的一类具有强力抗癌活性的天然产物。它们可以嵌入DNA的小沟中，在N3位置烷基化DNA中的腺嘌呤。荷兰Synthon公司的vc-seco-DUBA是在DNA抑制剂duocarmycin的基础上设计的连接头和小分子载荷的组合。使用这一组合的ADC分子SYD985(Anti-HER2-vc-seco-DUBA)在临床前和临床上都显示了优于T-DM1的疗效。
SN-38是DNA拓扑异构酶I抑制剂伊立替康的活性代谢物，其抗癌活性和毒性都相 对较低。Immunomedics公司的sacituzumab govitecan(IMMU-132)就选用了SN-38并在临床上取得了不错的效果。另外一个在ADC中采用的DNA拓扑异构酶I抑制剂是PNU-159682，它是多柔比星类似物nemorubicin的活性代谢物，但是活性比多柔比星提高了3000倍。NBE Therapeutics公司是采用这一载荷的先驱者。
在选择小分子载荷时，还要考虑是否是多药耐受蛋白(multiple drug resistance protein，MDR)，比如MDR1(又称P-glycoprotein)的底物。多药耐受蛋白是多个表达在细胞膜上的糖蛋白，它们可以把MMAE和DM1等小分子化合物泵出肿瘤细胞，从而使细胞获得耐药性。化疗可以上调多药耐受蛋白在病人的肿瘤组织中的表达量，因此选择非多药耐受蛋白底物的小分子药物如多卡霉素和PNU-159682等可以部分克服因这类蛋白上调带来的耐药性。
L为连接头，把抗体和载荷两个功能模块通过共价偶联的方式连接到一起。抗体分子自身的半胱氨酸或赖氨酸残基可以作为连接点。偶联方式可以是非定点方式，即最终的产物是在不同的位点偶联了不同数目的载荷分子的混合物。现在已经上市的两个ADC分子都是通过非定点方式偶联的。定点偶联是通过在抗体分子上添加人工半胱氨酸、糖链修饰、掺入非天然氨基酸或通过酶学催化修饰(如转肽酶和转胺酶修饰)等方法来精确控制载荷分子添加的位点和数目。
ADC分子中载荷的高效率释放是影响ADC分子活性的一个重要因素。而这一过程是通过断裂抗体和载荷之间的化学连接头实现的，所以选择适当的连接头、在适当的位点偶联适当个数的载荷，是ADC发挥疗效的关键。一般的，理想的连接头要在外周血液循环中足够稳定，从而避免因细胞毒分子释放产生的毒性。同时，ADC被内吞进入细胞后，要能够有效地释放载荷。
ADC分子的连接头从性能上可分为可裂解的和不可裂解的两类。可裂解的连接头又包括化学裂解和酶催化裂解两种。目前常用的化学裂解连接头有酸不稳定腙键连接头和二硫键连接头：二肽连接头属于酶催化裂解连接头；硫醚键和酰胺键属于不可裂解的连接头。
酸不稳定腙键连接头在溶酶体等酸性条件下不稳定，而在外周循环系统中性条件下较为稳定。其被应用于抗体-刺孢霉素偶联的ADC中，部分腙键连接头在外周循环系统中24小时约有5％-6％的细胞毒分子脱落，而在癌细胞内可高达97％-98％。但是，腙键连接头在循环系统中的半衰期仍较短(43小时)，而很多裸抗体本身在人体内的半衰期可达数天甚至数周。因此，其选择性仍相对较低。二硫键也是目前ADC常用的连接头之一。它在循环系统中稳定，在胞内还原条件下被裂解，从而释放出载荷。
二肽连接头如缬氨酸-瓜氨酸(valine-citrulline，vc)，可以被特定的溶酶体蛋白酶裂解，比化学裂解性连接头在人类循环系统的稳定性高100倍，在小鼠和猴体内的半衰期约为6-10天。与其他二肽相比，缬氨酸-瓜氨酸在血液中表现出相对较好的稳定性，在细胞溶酶体内的组织蛋白酶B等催化作用下更容易被裂解，因此是目前应用最广泛的ADC肽链连接头之一。
硫醚连接头是不可裂解的稳定的连接头。应用此类连接头的ADC被肿瘤细胞内化以后，抗体部分在溶酶体内被降解，释放出带有硫醚连接头的小分子细胞毒分子，发挥抗肿瘤效应。采用硫醚键可以提高ADC分子在血液循环和细胞内环境中的稳定性和耐受性，不过其释放小分子毒素的速率较慢，并会与人血清白蛋白发生巯基交换，从而限制 了其临床应用。
肿瘤相关的钙信号传导者2(Tumor associated calcium signal transducer 2，TACSTD2)，又称Trop-2、EGP-1、MS-1、Gp-15、T-16和GA733-I，是一个分子量为45kDa的呈单体形式的一型跨膜糖蛋白。根据基因序列推测，它的蛋白序列含有信号肽(26个氨基酸残基)、胞外区(248个氨基酸残基)、跨膜区(23个氨基酸残基)和胞内区(26个氨基酸残基)。
在多种上皮源性的实体肿瘤(如乳腺癌、宫颈癌、结直肠癌、肾癌、肝癌、肺癌、胰腺癌和前列腺癌等)组织中，TACSTD2均高比例地过量表达。比如有76％的乳腺癌、55％的胰腺癌和56％的胃癌病人的病灶细胞过量表达该抗原。临床资料表明TACSTD2的表达水平与肿瘤的侵袭性呈正相关，而与患者的预后呈负相关。在癌变细胞上，TACSTD2是一个具有信号传导功能的致癌基因。它可能通过抗体交联或胞内片段部分的剪切而被激活。激活以后，它的胞内区被磷酸激酶C磷酸化，并介导钙信号的传递。钙信号可以激活或上调多种促进细胞分裂和生长的信号传导分子，如NFκB、cyclin D-1和ERKs等，从而加速细胞的生长、侵袭和转移。另外，还有大量的TACSTD2表达在癌变细胞内部，表达量因细胞种类的不同而差异很大，可能是一种对TACSTD2功能的调控手段。TACSTD2在肿瘤细胞与正常细胞之间表达量和功能的巨大差异使它成为一个理想的药物作用靶点。
本领域技术人员致力于开发新的、更有效的靶向TACSTD2的抗体与药物偶联体，并发现靶向TACSTD2的人源化抗体hRS7与MMAE搭配时具有良好的协同作用，达到了非常突出的药效。
大多数ADC分子的Ab具有亲水性，而小分子毒素D则一般采用疏水性化合物。这种分子两端极性的差异极易造成ADC分子的聚集，从而影响其成药性和药效。本发明找到一种通过寡聚PEG来改良vc二肽连接头的特定方法，该方法可以平衡小分子毒素D的疏水性，从而改善ADC的分子整体的成药性和药效，可以用于多种不同的ADC分子的偶联。
发明内容
本发明的目的在于(1)提供一种靶向TACSTD2的抗体与药物偶联体(ADC)分子，该分子为抗体hRS7与MMAE的特定组合，该组合具有明显的协同作用；(2)提供一种优化的ADC分子的二肽连接头技术。
在本发明的第一方面中，提供了一种抗体与药物偶联体(ADC)或其药学上可接受的盐，所述抗体与药物偶联体结构如式I所示：
Ab-(L-D)n      I
其中，
Ab表示抗体，且Ab为hRS7：
D为具有细胞毒性的小分子药物，所述小分子药物选自下组：单甲基澳瑞他汀(monomethyl auristatin)、加利车霉素、美登素类、多柔比星(阿霉素)、吡咯苯并二氮杂卓(pyrrolobenzodiazepine)(PBD)、多卡霉素(duocarmycin)，或其组合；
L为连接所述抗体和所述药物的接头；
n为偶联于所述抗体的所述药物的平均偶联数量，可以是整数或非整数的正数，且0.8≤n≤8；
“-”为键或接头。
在另一优选例中，式I中，n为偶联于所述抗体的所述药物的平均偶联数量，也是药物与抗体的比率(drug antibody ratio)，又称DAR值。
在另一优选例中，n为1-7的整数或非整数的正数。
在另一优选例中，n为1-6的整数或非整数的正数：较佳地，为2-6的整数或非整数的正数；更佳地，为2-4的整数或非整数的正数。
在另一优选例中，D为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)、多柔比星、吡咯苯并二氮杂卓、多卡霉素，或其组合。
在另一优选例中，Ab为人源化的靶向TACSTD2的抗体hRS7。
在另一优选例中，D为具有细胞毒性的小分子药物单甲基澳瑞他汀-E(MMAE)。
在另一优选例中，L为所以可以有效地共价偶联hRS7与MMAE的连接头。
在另一优选例中，n为1-6的正数：较佳地，为2-6的正数：更佳地，为2-4的正数。
在另一优选例中，D为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)或单甲基澳瑞他汀-F(MMAF)。
在另一优选例中，L为mc(maleimidocaproxyl，马来酰亚胺己酰基)-vc(valine-citrulline，缬氨酸-瓜氨酸)-PABC(para-aminobenzyloxycarbonyl，对氨基苄氧羰基)。
在另一优选例中，L为MCC(maleimidomethyl cyclohexane-I-carboxylate，马来酰亚胺甲基环己基-I-羧酸酯基)-vc-PABC。
在另一优选例中，vc连接头被CL2A代替。
在另一优选例中，L为mc-vc-PABC，D为MMAE时，所述的抗体与药物偶联体为hRS7-[mc-vc-PABC-MMAE]n(可简略为hRS7-[vc-MMAE]n或hRS7-vc-MMAE)，其中，n为正数，其结构如式II所示：
Figure PCTCN2017000685-appb-000001
在另一优选例中，L中包含x个寡聚的聚乙二醇PEG(可用PEGx表示，其中，x为聚乙二醇PEG的重复个数)，且x为大于零的整数。
在另一优选例中，当L中包含x个寡聚的聚乙二醇PEG时，所述抗体与药物偶联体可以表示为Ab-(L(PEGx)-D)n。
在另一优选例中，所述PEGx可选自下组的一个或多个：PEG2、PEG4、PEG6，PEG8、PEG12、PEG16、PEG24、PEG28等，其中，PEG后的数字代表PEG的重复个数。
在另一优选例中，所述抗体与药物偶联体为hRS7-[L(PEGx)-MMAE]n，其中x为PEG的重复个数，优选为1-28的整数：较佳地，为4-24的整数。
在另一优选例中，所述抗体与药物偶联体为hRS7-[mc-PEGx-vc-PABC-MMAE]n(可简略为hRS7-vc(PEGx)-MMAE)，其中x为PEG的重复个数，优选为1-64的整数；较佳地，为4-24的整数。
在另一优选例中，PEG可以与马来酸连接。
在另一优选例中，所述抗体与药物偶联体(ADC)如式III或IV所示：
Figure PCTCN2017000685-appb-000002
Figure PCTCN2017000685-appb-000003
在另一优选例中，L中可以嵌入更多的聚乙二醇(PEGx)以改善分子的亲水性质，所述抗体与药物偶联体(ADC)如式V-1、V-2、V-3所示：
Figure PCTCN2017000685-appb-000004
Figure PCTCN2017000685-appb-000005
Figure PCTCN2017000685-appb-000006
在V-2所示的优选例中，L中的瓜氨酸(citrulline，C)残基被赖氨酸、精氨酸(argine)或天冬氨酸(glutamic acid)残基所替代，然后将寡聚PEG24连接到该氨基酸侧链的活性基团上，采用该连接方式的分子显示了最优的成药性和药效；
在另一优选例中，式II中，当L中的缬氨酸(valine，V)残基被带有活性基团的氨基酸残基如赖氨酸(lysine)、精氨酸(Argine)或天冬氨酸(glutamic acid)残基所替代，然后将PEGx连接到该氨基酸侧链的活性基团上，所述抗体与药物偶联体如结构式VI所示，其中x为1-28的整数：n为1-8的正数；
Figure PCTCN2017000685-appb-000007
在另一优选例中，所述抗体hRS7具有以下特征：
(a)所述抗体hRS7的轻链可变区的氨基酸序列包含或为如SEQ ID NO.：1所示的序列；和/或
(b)所述抗体hRS7的重链可变区的氨基酸序列包含或为如SEQ ID NO.：2所示的序列。
本发明第二方面提供了一种药物组合物，所述药物组合物包含本发明第一方面所述的抗体与药物偶联体，以及药学上可接受的载体。
本发明第三方面提供了本发明第一方面所述的抗体与药物偶联体或本发明第二方面所述的药物组合物的用途，用于制备抗肿瘤的药物。
在另一优选例中，所述肿瘤包括但是不限于乳腺癌(三阴及非三阴)、胃癌、卵巢癌、小细胞和非小细胞(鳞癌、腺癌、腺鳞癌、大细胞癌)肺癌、宫颈癌、子宫癌，食道癌、头颈癌、胰腺癌、大肠癌、膀胱癌、肝癌、前列腺癌以及其它高表达TACSTD2的实体肿瘤。
本发明第四方面提供了一种制备本发明第一方面所述的抗体与药物偶联体的方法，所述方法包括步骤；
(1)提供一反应体系，所述反应体系中包括抗体和药物分子，所述药物分子连接有接头；
(2)在所述反应体系中，将所述抗体和药物分子进行偶联反应，从而制得所述的抗体与药物偶联体。
在另一优选例中，所述反应体系的pH为6.0-8.0；较佳地，为6.0-7.0。
在另一优选例中，所述抗体与药物分子的摩尔比为1∶1～1∶20；较佳地，为1∶10～1∶20。
在另一优选例中，反应时间为1h～48h；较佳地，为3h～36h。
本发明第五方面提供了一种治疗或预防肿瘤的方法，所述方法包括步骤：给需要的对象施用本发明第一方面所述的抗体与药物偶联体或本发明第二方面所述的药 物组合物。
在另一优选例中，所述对象为哺乳动物，包括人。
在另一优选例中，所述抗体与药物偶联体的给药浓度为0.3-10毫克/千克；较佳地，为1-6毫克/千克；更佳地，为1-4毫克/千克。
本发明的第六方面提供了一种通用的PEG化的二肽连接头技术。具体地，提供了一种用于连接小分子药物和生物大分子的连接头L1，L1含有类似于vc(valine-citrulline，缬氨酸-瓜氨酸)的可以被蛋白酶B切断结构的ADC连接头，其中，瓜氨酸残基被赖氨酸残基所代替，且赖氨酸残基的活性氨基可以连接x个寡聚的聚乙二醇PEG。
在另一优选例中，L1含有vc(valine-citrulline，缬氨酸-瓜氨酸)，其中，瓜氨酸残基被赖氨酸残基所代替，且赖氨酸残基的活性氨基可以连接x个寡聚的聚乙二醇PEG。其中，缬氨酸-瓜氨酸可以被蛋白酶B切断。
在另一优选例中，L1含有缬氨酸-赖氨酸(VL)。较佳地，赖氨酸残基的活性氨基可以连接x个寡聚的聚乙二醇PEG。
在另一优选例中，所述的x个寡聚的聚乙二醇PEG可用PEGx表示，其中，x为聚乙二醇PEG的重复个数，且为大于零的整数。
在另一优选例中，L1含有缬氨酸-赖氨酸(m-dPEGx)-PAB(其中PAB也可表示为PABC)。其中-为键或连接基团。
在另一优选例中，缬氨酸上连接有PEGx。其中PEG与缬氨酸通过羰基连接。
在另一优选例中，L1为PEGx-缬氨酸-赖氨酸(m-dPEGx)-PAB
在另一优选例中，L1为PEG4-VL(m-dPEG24)-PAB。采用该修饰方式的ADC分子具有最佳的成药性和体内药效。
在另一优选例中，L1还含有马来酰亚胺己酰基.
在另一优选例中，L1中，缬氨酸上还连接有马来酰亚胺己酰基。
在另一优选例中，当缬氨酸上连接有PEGx时，马来酰亚胺己酰基连接在PEGx上。例如L1为mc-PEG4-VL(m-dPEG24)-PAB。
在另一优选例中，所述生物大分子具有靶向功能，所述生物大分子包括抗体、融合蛋白等。
在另一优选例中，所述小分子药物具有细胞毒性，所述小分子药物包括但不限于：单甲基澳瑞他汀(monomethyl auristatin E)-E、-D或-F(MMAE、MMAD或MMAF)、加利车霉素、美登素类(DM1和DM4等)、多柔比星(阿霉素)、吡咯苯并二氮杂卓(pyrrolobenzodiazepine，PBD)、多卡霉素(duocarmycin)，奈莫柔比星(nemorubicin)类似物(如PNU-159682)，或两种以上小分子药物的组合。
在另一优选例中，所述连接头连接小分子药物和生物大分子的结构可以如式I-1所示：
Ab-(L1-D)n      I-1
其中，
Ab为任何具有靶向功能的生物大分子，所述生物大分子包括抗体、融合蛋白等：
D为具有细胞毒性的小分子药物，所述小分子药物包括但不限于：单甲基澳瑞他汀(monomethyl auristatin E)-E、-D或-F(MMAE、MMAD或MMAF)、加利车霉素、 美登素类(DM1和DM4等)、多柔比星(阿霉素)、吡咯苯并二氮杂卓(pyrrolobenzodiazepine，PBD)、多卡霉素(duocarmycin)，奈莫柔比星(nemorubicin)类似物(如PNU-159682)，或两种以上小分子药物的组合：
n为偶联于所述抗体的所述药物的平均偶联数量，可以是整数或非整数的正数，且0.8≤n≤8；
“-”为键或接头。
在另一优选例中，L1为mc(maleimidocaproxyl，马来酰亚胺己酰基)-vc(valine-citrulline，缬氨酸-瓜氨酸)-PABC(para-aminobenzyloxycarbonyl，对氨基苄氧羰基)。
在另一优选例中，L1为MCC(maleimidomethyl cyclohexane-I-carboxylate，马来酰亚胺甲基环己基-I-羧酸酯基)-vc-PABC。
在另一优选例中，vc连接头被CL2A代替。
在另一优选例中，L1为mc-vc-PABC，D为MMAE时，所述的抗体与药物偶联体为hRS7-[mc-vc-PABC-MMAE]n(可简略为hRS7-[vc-MMAE]n或hRS7-vc-MMAE)，其中，n为正数，其结构如式II所示：
Figure PCTCN2017000685-appb-000008
在另一优选例中，L1中包含x个寡聚的聚乙二醇PEG(可用PEGx表示，其中，x为聚乙二醇PEG的重复个数)，且x为大于零的整数。
在另一优选例中，当L1中包含x个寡聚的聚乙二醇PEG时，所述抗体与药物偶联体可以表示为Ab-(L1(PEGx)-D)n。
在另一优选例中，所述PEGx可选自下组的一个或多个：PEG2、PEG4、PEG6，PEG8、PEG12、PEG16、PEG24、PEG28等，其中，PEG后的数字代表PEG的重复个数。
在另一优选例中，L1中可以嵌入更多的聚乙二醇(PEGx)以改善分子的亲水性质，所述抗体与药物偶联体(ADC)如式V-1、V-2、V-3所示：
Figure PCTCN2017000685-appb-000009
Figure PCTCN2017000685-appb-000010
Figure PCTCN2017000685-appb-000011
在V-2所示的优选例中，L1中的瓜氨酸被赖氨酸、精氨酸(Argine)或天冬氨酸(glutamic-acid)残基所替代，然后将寡聚PEGx连接到该氨基酸侧链的活性基团上，采用该连接方式的分子显示了最优的成药性和药效。该连接头可以用于多种不同的ADC分子的偶联，包括但不限于靶向TACSTD2的ADC分子。
在另一优选例中，式II中，当L1中的缬氨酸(valine，V)残基被带有活性基团的氨基酸残基如赖氨酸(lysine)、精氨酸(Argine)或天冬氨酸(glutamic acid)残基所替代，然后将PEGx连接到该氨基酸侧链的活性基团上，所述抗体与药物偶联体如结构式VI所示，其中x为1-28的整数：n为1-8的正数：
Figure PCTCN2017000685-appb-000012
在另一优选例中，连接头L1可以是本文中所给出的任一ADC分子中的连接头或接头。
在另一优选例中，连接头L1可以是实施例11中所给出结构式中的连接头或接头中的一种或多种。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案，限于篇幅，在此不再一一累述。
附图说明
图1显示了hRS7-vc-MMAE在体外对胰腺癌细胞BxPC-3的杀伤效力显著高于hRS7-CL2A-SN38。尽管MMAE本身对肿瘤细胞的杀伤力要强于SN38，然而在与hRS7 配伍以后，这种增强的杀伤力被急剧的放大，显示抗体hRS7与MMAE具有明显的协同作用。
图2显示了hRS7-vc-MMAE在移植了1x106、2x106、5x106BxPC3的BALB/c-Nu肿瘤裸鼠模型上显示强力的药效趋势。Rx：3毫克/千克，QW，i.v.。肿瘤体积＝长×宽2/2。
图3显示了hRS7-vc-MMAE在移植了MBA-MB-468的BALB/c-Nu肿瘤裸鼠模型上显示强力的药效。QW，i.v.。肿瘤体积＝长×宽2/2。n＝5。其中1和3毫克/千克剂量组药效显著，而0.3毫克/千克剂量组则效果不佳。
图4显示了不同DAR值的hRS7-vc-MMAE在乳腺癌细胞MBA-MB-468上的杀伤效率。其中DAR值为2，4和6的分子药效相似，而当DAR值低于2时，细胞杀伤效果不佳。
图5显示了在接头(或连接链)中嵌入PEG4的ADC分子hRS7-mc-PEG4-vc-PABC-MMAE(简略为hRS7-vc(pEG4)-MMAE)具有更加显著的药效。
图6显示了各个ADC分子的稳定性实验结果。
图7显示了各个ADC分子的稳定性实验结果。
图8显示了各个ADC分子的分子疏水性。
图9显示了各个ADC分子的分子疏水性。
图10显示了各个ADC分子的细胞杀伤活性。
图11显示了不同PEG修饰Linker的ADC对小鼠接种MDA-MB-468肿瘤的抑制，其中hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE的肿瘤抑制效果量佳。
图12显示了PEG4-VL(m-dPEG24)-PAB连接头可以用于偶联不同的小分子毒素。
具体实施方式
本发明人通过广泛而深入的研究，提供大量筛选，获得一种高效的，负载量、稳定性和疏水性经过优化的，靶向TACSTD2的抗体与药物偶联体。实验结果表明，本发明的抗体与药物偶联体通过将抗体hRS7与小分子药物(如MMAE)的合理配伍，从而取得了异常显著的抗肿瘤效果；另一方面，采用优化的负载量，显著降低了本发明抗体与药物偶联体对其它细胞(如正常细胞)的毒副作用。另外，在偶联体的二肽连接头中的瓜氨酸位置嵌入聚乙二醇，进一步改善分子的理化性质，提高药效，该连接头技术可以用于多种ADC分子的偶联。在此基础上完成了本发明。
定义
如本文所用，术语“本发明的抗体与药物偶联体”、“本发明的抗体与药物偶联物”、“本发明的偶联体”或“本发明的ADC”可互换使用，指具有式I结构所示的抗体-药物偶联体。另外，本发明中，同一种或同一类ADC分子可以用多种形式表示。
例如，其中一类ADC分子，hRS7-[mc-vc-PABC-MMAE]n，当n为不同数值时，可以通称为hRS7-vc-PAB-MMAE或hRS7-mc-vc-PABC-MMAE或hRS7-vc-MMAE或hRS7-mal-vc-PABC-MMAE。hRS7-[mc-vc-PABC-MMAE]n中的n数值即为ADC分子的DAR值。例如，在上下文中，DAR为2的hRS7-mc-vc-PABC-MMAE或 hRS7-vc-MMAE，即hRS7-[mc-vc-PABC-MMAE]2。
又例如，hRS7-vc(PEG4)-MMAE可以表示为hRS7-mc-PEG4-vc-PABC-MMAE或hRS7-PEG4-vc-PAB-MMAE。
又例如，Mal-Peg4-Val-Lys(m-dPEG24)-PAB-MMAE也可表示为Mal-Peg4-Val-Lys(PEG24-Me)-PABC-MMAE，尤其和hRS7形成的ADC分子可表示为hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE；
Mal-Peg4-Lys(Peg24-Me)-Cit-PAB-MMAE也可表示为Mal-Peg4-Lys(PEG24-Me)-Cit-PABC-MMAE，尤其和hRS7形成的ADC分子可表示为hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE；
Mal-PEG24-VC-PAB-MMAE也可表示为Mal-PEG24-Val-Cit-PABC-MMAE，尤其和hRS7形成的ADC分子可表示为hRS7-PEG24-vc-PAB-MMAE。
PEGx也可以表示为dPEGx。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、994等)。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区：轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
在特殊情况下，ADC分子中的抗体部分可以被具有靶向作用的融合蛋白代替。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单 位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段， 或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等，Anal.Biochem.，107：220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
在另一优选例中，所述靶向TACSTD2的抗体为抗体hRS7。
在另一优选例中，所述抗体hRS7的轻链可变区(V-Kappa)氨基酸序列为如SEQ ID NO.：1所示的氨基酸序列(DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ)。
在另一优选例中，所述抗体hRS7的重链可变区(VH)氨基酸序列为如SEQ ID NO..2所示的氨基酸序列(QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS)。
小分子药物
适用于本发明的小分子药物为具有高细胞毒性的化合物，优选单甲基澳瑞他汀(monomethylauristatin)、加利车霉素、美登素类、多柔比星(阿霉素)、吡咯苯并二氮杂卓(pyrrolobenzodiazepine)(PBD)、多卡霉素(duocarmycin)，或其组合：更佳地选自：单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)或其组合。
接头或连接头(L或L1)
适用于本发明的接头(L或L1)用于连接本发明的小分子药物和抗体。
在另一优选例中，L或L1为mc-vc-PABC(可简称为vc)。
在另一优选例中，L或L1为MCC-vc-PABC。
本发明的L或L1中可以包含x个寡聚的聚乙二醇(可用PEGx表示，其中，x为聚乙二醇(PEG)的重复个数)。其中，x可以为1-28的整数。
在另一优选例中，所述PEGx可选自下组的一个或多个：PEG2、PEG4、PEG6，PEG8、PEG12、PEG16、PEG24、PEG28等，其中，PEG后的数字代表PEG的重复个数。
在另一优选例中，PEG可以与马来酸连接。
抗体与药物偶联体及其制备
本发明提供了一种抗体与药物偶联体(ADC)，它包括通过接头(L或L1)连接在一起的(a)靶向TACSTD2的抗体与(b)具有细胞毒性的小分子药物。
本发明提供了偶联方法，将小分子药物通过特定连接物偶联到抗体上，在不改变抗体亲和性的基础上大幅提高抗体对肿瘤细胞的杀伤力。
典型的适用于本发明的偶联方式，包括K-Lock和C-Lock两种偶联方式。在K-Lock偶联方式中，药物分子偶联于抗体序列中赖氨酸(K)残基，在C-Lock偶联方式中，药物分子偶联于抗体序列中的半胱氨酸(C)残基。
在另一优选例中，本发明采用一种特定的接头(L或L1)部分，使之能够偶联至特定的赖氨酸上，从而获得定点偶联的、且负载量优化的抗体偶联药物。
在另一优选例中，本发明的抗体与药物偶联体的制备方法如下：
吸取一定量(如0.5毫克)的抗体，加入2倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)，37℃条件下反应一段时间(如2.5小时)。反应完毕以后，我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体10倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon微升Tra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。
在另一优选例中，本发明的抗体与药物偶联体的制备方法如下：
吸取一定量(如0.5毫克)的抗体，加入3倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)，37℃条件下反应一段时间(如2.5小时)。然后直接加入抗体20倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。
在另一优选例中，本发明的抗体与药物偶联体的制备方法如下：
吸取一定量(如0.5毫克)的抗体，加入3倍物质当量的二硫苏糖醇(DTT)，37℃条件下反应一段时间(如2小时)。反应完毕以后，我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体10倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。
在另一优选例中，L或L1为mc-vc-PABC，D为MMAE时，所述的抗体与药物 偶联体为hRS7-[mc-vc-PABC-MMAE]n(可简略为hRS7-[vc-MMAE]n或hRS7-vc-MMAE)，其中，n为1-8的正数，其结构如式II所示；
Figure PCTCN2017000685-appb-000013
在另一优选例中，当L或L1中包含x个寡聚的聚乙二醇PEG时，所述抗体与药物偶联体可以表示为Ab-(L(或L1)(PEGx)-D)n。
在另一优选例中，所述抗体与药物偶联体为hRS7-[L(或L1)(PEGx)-MMAE]n，其中x为PEG的重复个数，优选为1-28的整数；较佳地，为4-24的整数。
在另一优选例中，所述抗体与药物偶联体为hRS7-[mc-PEGx-vc-PABC-MMAE]n(可简略为hRS7-vc(PEGx)-MMAE)，其中x为PEG的重复个数，优选为1-64的整数；较佳地，为4-24的整数。
在另一优选例中，所述抗体与药物偶联体(ADC)如式III或IV所示：
Figure PCTCN2017000685-appb-000014
在另一优选例中，L(或L1)中可以嵌入更多聚的聚乙二醇(PEG)以改善分子的亲水性质，所述抗体与药物偶联体(ADC)如式V-1、V-2、V-3所示：
Figure PCTCN2017000685-appb-000016
Figure PCTCN2017000685-appb-000017
Figure PCTCN2017000685-appb-000018
在另一优选例中，式II中，当L(或L1)中的缬氨酸残基被带有活性基团的氨基酸残基如精氨酸或天冬氨酸所替代，然后将PEGx连接到该氨基酸侧链的活性基团上，所述抗体与药物偶联体如结构式VI所示，其中x为1-28的整数；n为1-8的正数；
Figure PCTCN2017000685-appb-000019
药物组合物和施用方法
本发明还提供了含有本发明ADC的药物组合物，以及使用本发明ADC治疗哺乳动物疾病的方法。优选地，所述的疾病是以TACSTD2为靶向的疾病，如多种恶性实体肿瘤。
本发明还提供了所述抗体与药物偶联体在制备抗肿瘤药物中的应用。
在本发明中，所述药物组合物包括有效量的根据本发明的ADC(作为活性成分)，以及至少一种药学上可接受的载体、稀释剂或赋形剂。制备时，通常将活性成分与赋形剂混合，或用赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可采用固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此，组合物可以是溶液剂、灭菌注射溶液等。
合适的赋形剂包括：乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水等；制剂还可包括：湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)等。所述抗肿瘤药物可制成单元或多元剂型，各剂型包含为了产生所期望的疗效而计算出预定量的本发明的ADC，以及合适的药剂学赋形剂。
所述的抗肿瘤药物可以通过常规途径进行给药，包括(但并不限于)：肌内、腹膜内、静脉内、皮下、皮内、局部给药等。
使用该药物时，是将安全有效量的所述抗体与药物偶联体施用于人，其中该安全有效量的范围优选为0.5～50毫克/千克体重，更优选为1～10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。
此外，本发明的偶联体还可与其他治疗药物联用，其中包括(但并不限于)：各种细胞因子，如TNF、IFN、IL-2等；各种肿瘤化疗药物，如5-FU、氨甲喋呤等影响核酸生物合成的药物；氮芥、环磷酰胺等烷化剂类药物；阿霉素、放线菌素D等干扰转录过程阻止RNA合成的药物：长春新碱、喜树碱类等影响蛋白质合成的药物及某些激素类药物，等等。
与现有技术相比，本发明的主要有益效果包括：
(1)本发明提供的抗体与药物偶联体对肿瘤细胞具有非常突出的杀灭肿瘤细胞的的活性，抗体与小分子毒素的协同作用明显。
(2)本发明提供的抗体与药物偶联体具有经优化的DAR值和更优的载荷，使得本发明ADC的治疗窗口更大。
(3)本发明提供的抗体与药物偶联体具有经优化的DAR值2-4，与高DAR值的ADC相比，更多地保留了抗体中的二硫键，使得本发明ADC具有更高的稳定性。
(4)本发明在连接链的瓜氨酸部位中引入多聚的聚乙二醇，从而显著改善了分子的理化性质，提高了药效。
(5)本发明提供的抗体与药物偶联体具有更好的药代性能以及更低的毒副作用。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆；实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
材料和通用方法
isotype指同型对照抗体，此处为人源化的靶向ToxinB的抗体，属于人IgGI抗体类型。
MDA-MB-468细胞为常规的三阴乳腺癌细胞，购自中科院上海细胞库。
BxPC-3为常规的胰腺癌细胞，可购自中科院上海细胞库。
HEK293F细胞购自赛默飞中国有限公司。
CHO-K1细胞购自美国ATCC。
mc-vc-PABC-MMAE购自南京联宁生物医药科技有限公司。
hRS7-CL2A-SN38：制备方法参见专利文献US 8,758,752 B2。
实施例1 抗体与药物偶联体制备
I、制备抗体蛋白溶液：
利用分子克隆技术，首先将hRS7抗体的轻链(氨基酸序列如SEQ ID NO：1所示)和重链(氨基酸序列如SEQ ID NO.：2所示)克隆到表达载体pcDNA3.1上。然后在大肠杆菌中扩增出分别带有抗体轻链和重链的cDNA序列的质粒，随后用转染试剂PEI转染在无血清培养基中悬浮培养的HEK293或CHO细胞，并在37℃、5％CO2条件下震荡培养4-10天。在GE AKTA Purifiier上使用Protein A和Superdex 200分子排阻层析柱对细胞培养上清中的抗体进行分离纯化，最后通过超滤浓缩得到1毫克/毫升以上浓度的抗体蛋白溶液。
II、制备抗体与药物偶联体；
如下实验可以通过改变还原以及偶联的条件，制备出不同DAR值的ADC分子hRS7-vc-MMAE(即hRS7-[mc-vc-PABC-MMAE]n，n即表示分子的DAR值)。下文中，vc-MMAE即mc-vc-PABC-MMAE。
(1)利用超滤法在PBS(pH7.0)缓冲液中将抗体浓缩至5-10毫克/毫升。按实测浓度计算，吸取1毫克抗体，加入1.5倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)37℃条件下反应2.5小时。反应完毕以后，我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体10倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。用此方法制备的ADC分子hRS7-vc-MMAE(1)，DAR值约为2。
(2)利用超滤法在PBS(pH7.0)缓冲液中将抗体浓缩至5-10毫克/毫升。按实测浓度计算，吸取1毫克抗体，加入10倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)37℃条件下反应2.5小时。反应完毕以后，我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体20倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。用此方法制备的ADC分子hRS7-vc-MMAE(2)，DAR值约为4。
(3)利用超滤法在PBS(pH7.0)缓冲液中将抗体浓缩至5-10毫克/毫升。按实测浓度计算，吸取1毫克抗体，加入25倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)37℃条件下反应2.5小时。反应完毕以后，我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体20倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。用此方法制备的 ADC分子hRS7-vc-MMAE（3)，DAR值约为6。
(4)利用超滤法在PBS(pH7.0)缓冲液中将抗体浓缩至5-10毫克/毫升。按实测浓度计算，吸取1毫克抗体，加入0.5倍物质当量的三(2-羧乙基)膦(Tris(2-carboxyethyl)phosphine，TCEP)37℃条件下反应2.5小时。反应完毕以后·我们通过离心超滤法把反应混合物浓缩到0.5毫升以下，并用偶联溶液(75mM NaAc，pH6.5，1mM DTPA，10％DMSO)加满，再离心浓缩到0.5毫升以下，重复三次。我们然后加入抗体5倍物质当量的vc-MMAE，4℃过夜反应(17小时以上)。我们最后通过Amicon Ultra 4 10K超滤管把缓冲液置换成pH7.0的PBS溶液。用此方法制各的ADC分子hRS7-vc-MMAE(4)，DAR值约为0.5。
(5)参照上述方法制各ADC分子hRS7-vc-MMAE(5)，DAR值约为2.8，不同点在于加入抗体的6倍物质当量的TCEP还原，加入10倍物质当量的vc-MMAE。
对照ADC分子的制备
isotypo-vc-MMAE，DAR值约为2.8，制各方法参照实施例1步骤II的实验(5)，不同之处在于使用hRS7同型对照抗体(人源的toxin B抗体)代替hRS7。
实施例2 肿瘤细胞抑制实验
实验步骤如下：
在96孔的细胞培养板中种入大约1-2万个细胞/100微升/孔(细胞数因肿瘤细胞系不同而异。胰腺癌细胞BxPC3是1万个细胞/孔，乳腺癌MDA-MB-468是2万/孔)。
药物分子按不同初始浓度用培养基精心1∶4等比稀释.每个浓度3个复孔加药(总体积200微升)。
给药组：hRS7-vc-MMAE(DAR值约为2.8)
对照组：hRS7-CL2A-SN38(DAR值约为2.8)
MMAE
SN-38
hRS7
isotype-vc-MMAE(DAR值约为2.8)
37℃培养96小时以后，加入10微升CCK8(细胞计数试剂盒-8)，继续培养1-4小时，然后测定在450nm的吸光度。我们用双尾的Student's t-test的统计方法来比较各给药组与对照组。
实验结果如下：对给药组和各个对照组在体外对胰腺癌细胞BxPC3的杀伤效力进行了初步的对比(结果如图1所示)。
由结果可知：MMAE比SN38更强力，两者的EC50值分别是0.27和6.9nM，相差20多倍。出入意料的是，当构建于以hRS7为抗体的ADC分子中时，MMAE与SN38的药效差别被急剧放大。hRS7-vc-MMAE杀伤BxPC3细胞的EC50值是0.27nM，而hRS7-CL2A-SN38则大于300nM。两者相比，hRS7-vc-MMAE的肿瘤细胞杀伤效力提高了1111倍(表1)。
表1.不同药物对BxPC3细胞杀伤效力的比较。
药物 EC50(nM)
MMAE 0.27
SN38 6.9
hRS7-vc-MMAE 0.27
hRS7-CL2A-SN38 ＞300
实验结果表明：从体外药效上看，MMAE与抗体hRS7构成ADC分子时具有显著的，但是难以预测的协同作用，即该协同作用是基于实验观察的，无法根据MMAE与SN38的药效不同而推测出来。
实施例3 体内抗肿瘤活性
实验步骤如下；
把胰腺癌细胞BxPC-3或乳腺癌细胞MDA-MB-468用生理盐水稀释到需要的细胞浓度，经皮下在4-6周年龄的雌性BALB/c-nu裸鼠的腋窝部位注射1-5x106个/300微升的肿瘤细胞。接种以后每天用游标卡尺测量肿瘤的尺寸。
肿瘤体积＝Lxω2/2
式中，L为最长的直径，ω是最短的直径。
当接种后36天或当肿瘤体积接近0.25cm3时，开始治疗实验。ADC分子一般每周注射一次(QW)，0.3-3毫克/千克体重。当肿瘤大于1.0cm3时，该小鼠被视为死亡。治疗效果可分为部分反应(肿瘤缩小30％或以上)，稳定(肿瘤缩小29％或以下，或增长不超过20％)。TTP(肿瘤增长时间)是从治疗开始到肿瘤增长20％体积的时间。对于肿瘤生长的统计分析是基于曲线下面积(AUC)。统计方法为双尾的Student’st-测验来比较各给药组与对照组。
给药组：ADC(hRS7-vc-MMAE，DAR值在胰腺癌和乳腺癌中分别约为2.8和4)
对照组：isotype-ADC(isotype-vc-MMAE，DAR值在胰腺癌和乳腺癌中分别约为2.8和4)
MMAE
hRS7
hRS7-CL2A-SN38(DAR值在胰腺癌和乳腺癌中分别约为2.8和4)
PBS
实验结果如下；
1)相对于isotype-vc-MMAE，hRS7-vc-MMAE在BxPC3细胞接种的胰腺癌模型上显示了非常强力的抑癌趋势。如图2所示。
2)相对于isotype-vc-MMAE，hRS7-vc-MMAE在MDA-MB-468细胞接种的三阴乳腺癌模型上显示了非常强力的抑癌效果，其中在1和3毫克/千克剂量组的药效明显，而0.3毫克/千克的药效不明显。如图3所示。
实验结果表明：hRS7-vc-MMAE在体内具有突出的肿瘤抑制活性。
实施例4 不同DAR值的ADC(hRS7-vc-MMAE)分子在MBA-MB-468细胞上的杀伤力比较
分别测试了实施例1制备的ADC分子的体内抗肿瘤活性。实验方法同实施例4，不同点在于，本次实验所用的肿瘤细胞为乳腺癌细胞MBA-MB-468。
实验结果如下：在MBA-MB-46细胞上，DAR值为2、4和6的ADC分子的肿瘤杀伤效力没有显著的区别，而DAR值低于2的ADC分子的药效较差。见图4。
结果表明：本发明制备的ADC分子在4以上具备优良的肿瘤杀伤效力。
实施例5 嵌入PEG4的hRS7-vc-MMAE分子在MBA-MB-468细胞上的杀伤力
hRS7-vc(PEG4)-MMAE(也称为hRS7-PEG4-vc-PAB-MMAE)的制备；
Mal-PEG4-NHS活化脂(26毫克，50umol，从Quanta Biodesign，USA购买)溶解于1毫升N.N-二甲基甲酰胺。在搅拌下，加入vc-PAB-MMAE(45毫克，40umol，从Levena Biopharma购买，按WO 2004/010957报道的方法合成)和9微升DIEA。反应液在室温(22℃)下搅拌一小时，反相高压液相制备纯化，冷冻干燥后得到白色固体产品Mal-PEG4-vc-PABC-MMAE(47毫克，78％).MS：m/z 1521.0(M+H+)。
将抗体与小分子Mal-PEG4-vc-PABC-MMAE偶联成为不同的DAR值的hRS7-vc(PEG4)-MMAE，偶联方法见实施例1。
通过MDA-MB-468肿瘤细胞抑制实验(实验方法见实施例4)，对hRS7-vc(PEG4)-MMAE和hRS7-vc-MMAE(两者的DAR值均为4)的药效进行了对比。
结果表明；在接头(或连接链)中嵌入PEG可以显著提高hRS7-vc-MMAE分子的药效。见图5。
实施例6 在连接链中引入24个聚乙二醇(PEG24)以及制备含有这些连接链的ADC分子的制备：
1)合成Mal-Peg4-Val-Lys(m-dPEG24)-PAB-MMAE
Figure PCTCN2017000685-appb-000020
Figure PCTCN2017000685-appb-000021
将Fmoc-Val-Lys(Trt)-PAB-PNP(100mg，0.102mmol，购买自Levena Biopharma，美国)和MMAE(78mg，0.102mmol，购买自Levena Biopharma，美国)溶解于无水DMF(5mL)，并在溶液中加入DIEA(7mg)。混合物在室温(22℃)下反应2小时，并通过反向HPLC直接纯化出目的产物Fmoc-Val-Lys(Trt)-PAB-MMAE(3)。产物(3)在冷冻干燥后形成为白色粉末(123mg，77％)。MS：m/z 1559.2(M+H+)。
将化合物3(120mg)溶于10％TFA/DCM(4mL)溶液并在室温下搅拌20分钟。混合物反应后通过减压浓缩和反向HPLC纯化，得到化合物4(88mg)。MS：m/z 1316.7(M+H+)。
将Fmoc-val-Lys-PAB-MMAE TFA盐(即化合物4)(80mg)，m-dPEG24酸(69mg)和HATU(23mg)溶于DMF(3ml)，加入DIEA(20mg)，并于室温下搅拌30分钟，获得所需产物[MS：m/z 1208.8(M/2+H+)]。将二异丙胺(1mL)加入到反应混合物中，并室温搅拌3小时。混合物反应后进行浓缩和反向HPLC纯化，在冻干后获得蜡状的化合物5(53mg)。MS：m/z 1097.3(M/2+H+)。
将化合物5(50mg)，Mal-PEG4-酸(10mg，购买自Quanta Biodesign，美国)和HATU溶于DMF(3mL)，加入DIEA(9mg)，并搅拌20分钟。之后通过反向HPLC纯化及冷冻干燥，最终获得所需化合物Mal-Peg4-Val-Lys(m-dPEG24)-PAB-MMAE化合物6(21mg)。MS：m/z 1296.1(M/2+H+)。
参照实施例1的方法，将抗体与化合物6偶联成ADC分子hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE。
2)合成Mal-Peg4-Lys(Peg24-Me)-Cit-PAB-MMAE
Figure PCTCN2017000685-appb-000022
将化合物1(80mg，购买自Levena Biopharma，美国)’MMAE(60mg，购买自Levena Biopharma，美国)，和DIEA(8mg)溶于干燥的DMF(3mL)，并于室温搅拌16小时。LC/MS检测反应产物生成[MS：m/z 1246.6(M+H+)]。在反应溶液中加入二异丙胺(1mL)并室温搅拌3小时。通过HPLC纯化并冻干获得白色粉末状的H-Cit-PAB-MMAE TFA盐(化台物2’)(92mg)。MS：m/z 1024.8(M+H+)。
将H-Cit-PAB-MMAE(即化合物2’)(40mg)，Fmoc-Lys(Boc)-OH(18mg，购买自Chem-Impex Inc.，美国)和HATU(15mg)溶于DMF(3mL)并加入DIEA(15mg)，于室温下搅拌20分钟。粗产物通过HPLC纯化成为所需产物Fmoc-Lys(Boc)-Cit-PAB-MMAE(41mg)。MS：m/z 1474.3(M+H+)。将生成的产物溶于10％TFA/DCM(5mL)，并于室温下搅拌20分钟。浓缩干燥获得粗产物，并进一步通过HPLC纯化分离获得白色粉末状的Fmoc-Lys-Cit-PAB-MMAE(31mg)(化合物3’)。MS：m/z 1374.2(M+H+)。
将化合物3’(31mg)，m-dPEG24(23mg，购买自Quanra Biodesign，美国)和HATU(8mg)溶于DMF(3ml)并加入DIEA(10mg)，于室温下搅拌20分钟。再加入二异丙胺(1mL)继续搅拌3小时。通过减压除去多余的碱，HPLC纯化后获得白色粉末状的H-Lys(m-dPEG24)-Cit-PAB-MMAE TFA盐(29mg)(化合物4’)。MS：m/z 1126.9(M/2+H+)。
将化合物4’(29mg)和Mal-PEG4-PFP(8mg，购买自Levena Biopharma，美国)溶于DMF(3mL)并加入DIEA(10mg)，于室温下搅拌10分钟。之后直接通过反向HPLC进行纯化，冻干后获得蜡状的目的分子Mal-PEG4-Lys(m-dPEG24)-Cit-PAB-MMAE(化合物5’)(19mg)。MS：m/z 1325.6(M/2+H+)。
参照实施例1的方法，将抗体与化合物5’偶联成ADC分子hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE。
3)合成Mal-PEG24-VC-PAB-MMAE
Figure PCTCN2017000685-appb-000023
将Mal-PEG24-NHS酯(化合物i)(70mg，50μmol，购买自Quanta Biodesign，美国)，vc-PAB-MMAE(化合物ii)(45mg，40μmol，购买自Levena Biopharma，依据WO 2004/010957发表的方法合成)和DIEA(9μL)溶于无水DMF(1mL)。将混合物在室温(22℃)下搅拌反应1小时，并通过反向HPLC直接进行纯化，冻干后获得无色浆状的目标产物Mal-PEG24-vc-PAB-MMAE(化合物iii)(70mg，73％)。MS：m/z 1202.1(M+2H+)/2。
参照实施例1的方法，将抗体与化合物iii偶联成ADC分子hR S7-PEG24-vc-PAB-MMAE。
实施例7 稳定性检测
由于hRS7-CL2A-SN38分子的DAR质偏高，导致分子的多个二硫键被打开，该分子的稳定性必然会受到影响。本发明制备的hRS7-vc-MMAE的DAR值更为合理，所以我们预测它的分子稳定性将更好，并使用了抗体稳定性试验验证这一假设。
我们将待储存于4℃的分析样品用室温下的PBS稀释至浓度为1毫克/毫升，取50μL直接进行测试(4℃储存条件)或放置于60℃水浴锅中孵育1小时(加速稳定性实验)之后再进行测试。我们在Thermo微升tiMate 3000 UHPLC高压液相仪上采用Thermo MAbPac SEC-1(7.8*300mm)分子排阻色谱柱对样品中的正常结构抗体或ADC分子(主峰)、聚合分子以及解链或降解的分子的含量进行定量分析。
UHPLC的流动相为50mM磷酸盐缓冲液(pH 7.0)+150mM NaCl，柱温25℃，进样体积为20μL，流速1毫升/min，UV检测波长214nm和280nm。我们最后通过Chromeleon软件进行数据采集和分析。
结果如图6所示，我们发现ADC分子的稳定性由于小分子的偶联而降低，与抗体hRS7(A与B)相比，出现更多的聚合和解链后的分子。其中，DAR＝6的分子在4℃储存条件下，有大量的解链或降解分子(I)，而在加速稳定性实验中则出现了大量的聚合体(J)，表明这个分子不够稳定。DAR＝4的ADC分子(E和F)与DAR＝2的ADC分子(C和D)相比，稳定性并没有显著的减低。另外，在4℃储存条件下，带有PEG4的ADC分子(G)的稳定性要优于不带PEG的ADC分子(E)，尽管在加速稳定性实验中两者的表现无显著差别。从图6的数据综合来看，DAR＝4，而连接链中加入PEG4的分子的稳定性最优。
接下来我们对在连接链中引入了PEG4和PEG24的ADC分子进行了稳定性的分析，hRS7-vc-MMAE和hRS7-PEG4-vc-PAB-MMAE作为对照。比较的分子包括：
1.hRS7-PEG24-vc-PAB-MMAE
2.hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE
3.hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE
4.hRS7-PEG4-vc-PAB-MMAE
5，hRS7-vc-PAB-MMAE(即hRS7-vc-MMAE)
如图7所示，在60℃条件下孵育1小时后，hRS7-vc-MMAE出现了51％的聚合体。PEG4的插入(hRS7-PEG4-vc-PAB-MMAE)使得聚集分子的部分降低到33％。然而，在连接链中直接加入PEG24后，分子出现了两个聚合峰，并且聚合分子比例占蛋白总量的45.1％，对分子的稳定性改善低于PEG4。然而当PEG24通过侧链连入ADC分子时，分子的稳定性得到了显著的改善，其中hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE的聚合比例降低到24.2％，hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE的聚合比例降低到27.2％。从图7的数据综合来看，hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE的分子的稳定性最优。
实施例8 疏水性检测
由于MMAE等小分子的疏水性非常高，导致分子的成药性较差。本发明在连接链中加入多个PEG以减低了分子的疏水性。PEG可以通过多种方式加入连接链。我 们使用了疏水柱，即hydrophobic interaction chromatography(HIC)，在HPLC上验证了这一假设并对PEG接入的方式进行了优化。
我们将待分析样品用色谱流动相A稀释至浓度为1毫克/千克，然后采用HIC进行样品疏水性分析。应用Agilent 1260 Infiinity HPLC进行检测，采用TOSOH TSKgel Butyl-NPR 4.6*35mm色谱柱，并通过Agilent OpenLAB软件进行数据采集分析。
色谱条件；流动相A为2M(NH4)2SO4 in 50mM磷酸缓冲液(pH 7.0)，流动相B为50mM磷酸缓冲液(pH 7.0)，梯度为0min 100％缓冲液A，5min 100％缓冲液A，25min 100％缓冲液B，30min 100％缓冲液B.柱温37℃，进样体积为10μL，流速1mL/min，UV检测波长214nm。
分子疏水性越高，在HIC柱中流出时间就越晚。在X轴上就越靠右。结果如图8的结果所示，不同DAR值的hRS7-vc-MMAE分子的疏水性随着DAR的增加而增加。由于DAR＝4与DAR＝6的药效相似，而DAR＝4的分子从疏水性上看优于DAR＝4，因此4是更优的DAR值选择。
图9中的数据显示，PEG化可以改变ADC分子的疏水性。其中hRS7-PEG4-vc-PAB-MMAE，hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE以及hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE的疏水性相对于hRS7-vc-PAB-MMAE都有降低。然而hRS7-PEG24-vc-PAB-MMAE对分子亲水性的改良效果不佳。
疏水性降低可以减少ADC分子在溶液中通过疏水的小分子部分相互作用而聚集的倾向。另外，疏水性的降低可以降低ADC分子被肝脏代谢排出的速度，从而延长分子在体内的半衰期、增强药效，并降低对肝脏的毒副作用。
实施例9 连接链的PEG化不影响ADC分子的肿瘤杀伤效率
我们对在连接链中引入了PEG4和PEG24的ADC分子进行了体外药效学比较，以确定PEG化不影响ADC分子的靶点识别、内吞、转运和载荷释放。
结果如图10和表2所示，在胰腺癌细胞BxPC3和乳腺癌细胞SK-BR-3上，hRS7-PEG24-vc-PAB-MMAE与hRS7-vc-MMAE相比的药效有明显的下降，可能与该分子较差的理化性质有关。)而hRS7-PEG4-vc-PAB-MMAE、hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE以及hRS7-PEG4-L(m-dPEG24)-C-PAB-MMAE与hRS7-vc-MMAE相比，细胞杀伤效率有提高或接近，表明PEG化不影响ADC分子的靶点识别、内吞、转运和载荷释放。
表2.不同PEG修饰的ADC分子对BxPC3和SK-BR-3的细胞杀伤效力(EC50值，nM)的比较。
Figure PCTCN2017000685-appb-000024
实施例10各PEG化分子的体内抗肿瘤活性比较
实验步骤如下：
把乳腺癌细胞MDA-MB-468用生理盐水稀释到需要的细胞浓度，经皮下在4-6周年龄的雌性BALB/c-nu裸鼠的腋窝部位注射1-5x106个/300微升的肿瘤细胞。接种以后每天用游标卡尺测量肿瘤的尺寸。
肿瘤体积＝Lxω2/2
式中，L为最长的直径，ω是最短的直径。
当接种后36天或当肿瘤体积接近0.25cm3时，开始治疗实验。ADC分子一般每周注射一次(QW)，0.3-3毫克/千克体重。当肿瘤大于1.0cm3时，该小鼠被视为死亡。治疗效果可分为部分反应(肿瘤缩小30％或以上)，稳定(肿瘤缩小29％或以下，或增长不超过20％)。TTP(肿瘤增长时间)是从治疗开始到肿瘤增长20％体积的时间。对于肿瘤生长的统计分析是基于曲线下面积(AUC)。统计方法为双尾的Student’st-测验来比较各给药组与对照组。
实验结果显示相对于其它PEG化分子，hRS7-PEG4-VL(m-dPEG24)-PAB-MMAE在体内具有更为突出的肿瘤抑制活性。
实施例11 PEG4-VL(m-dPEG24)-PAB连接头与不同小分子毒素的偶联
下图中的化合物名称1；SN-38；2；MMAF；3；DM1；4：活化的Duocarmycin TM：5：活化的PNU-159682
Figure PCTCN2017000685-appb-000025
Figure PCTCN2017000685-appb-000026
合成方法：将0.05mmol胺溶解于0.3mL无水DMF，加入10mg PNP碳酸酯(溶于0.3mL DMF)，再加入DIEA，调节pH到7。将混合物于室温搅拌16小时后，结束反应，并通过LC/MS确认。反应产物通过制备型反相HPLC进行纯化，最终获得浆状的产物。
Mal-PEG4-Val-Lys(m-dPEG24)-PAB-SN-38
Figure PCTCN2017000685-appb-000027
Mal-PEG4-Val-Lys(m-dPEG24)-PAB-DM1
Figure PCTCN2017000685-appb-000028
Mal-PEG4-Val-Lys(m-dPEG24)-PAB-MMAF
Figure PCTCN2017000685-appb-000029
Mal-PEG24-Val-Ala-PBD二聚体
Figure PCTCN2017000685-appb-000030
Mal-PEG4-Val-Lys(m-dPEG24)-PAB-DMEA-Duocarmycin TM
Figure PCTCN2017000685-appb-000031
Mal-PEG4-Val-Lys(m-dPEG24)-PAB-DMEA-PNU159682
Figure PCTCN2017000685-appb-000032
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种抗体与药物偶联体(ADC)或其药学上可接受的盐，其特征在于，所述抗体与药物偶联体结构如式I所示：
    Ab-(L-D)n           I
    其中，
    Ab表示抗体，且Ab为hRS7；
    D为具有细胞毒性的小分子药物，所述小分子药物选自下组；单甲基澳瑞他汀(monomethyl auristatin)、加利车霉素、美登素类、多柔比星(阿霉素)、吡咯苯并二氮杂卓(pyrrolohenzodiazepine)(PBD)、多卡霉素(duocarmycin)，或其组合；
    L为连接所述抗体和所述药物的接头；
    n为偶联于所述抗体的所述药物的平均偶联数量，可以是整数或非整数的正数，且0.8≤n≤8；
    “-”为键或接头。
  2. 如权利要求1所述的抗体与药物偶联体，其特征在于，n为1-7的整数或非整数的正数。
  3. 如权利要求I所述的抗体与药物偶联体，其特征在于，D为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)、多柔比星、吡咯苯并二氮杂卓、多卡霉素，或其组合。
  4. 如权利要求1所述的抗体与药物偶联体，其特征在于，L为mc(maleimidocaproxyl，马来酰亚胺己酰基)-vc(valine-citrulline，缬氨酸-瓜氨酸)-PABC(para-aminobenzyloxycarbonyl，对氨基苄氧羰基)。
  5. 如权利要求1所述的抗体与药物偶联体，其特征在于，L中包含x个寡聚的聚乙二醇PEG(可用PEGx表示，其中，x为聚乙二醇PEG的重复个数)，且x为1-28的整数。
  6. 如权利要求1所述的抗体与药物偶联体，其特征在于，所述抗体hRS7具有以下特征：
    (a)所述抗体hRS7的轻链可变区的氨基酸序列包含或为如SEQ ID NO.：1所示的序列：和/或
    (b)所述抗体hRS7的重链可变区的氨基酸序列包含或为如SEQ ID NO.：2所示的序列。
  7. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-6任一项所述的抗体与药物偶联体，以及药学上可接受的载体。
  8. 如权利要求1-6任一项所述的抗体与药物偶联体或如权利要求7所述的药物组合物的用途，其特征在于，用于制备抗肿瘤的药物。
  9. 一种制备权利要求1所述的抗体与药物偶联体的方法，其特征在于，所述方法包括步骤：
    (1)提供一反应体系，所述反应体系中包括抗体和药物分子，所述药物分 子连接有接头；
    (2)在所述反应体系中，将所述抗体和药物分子进行偶联反应，从而制得权利要求1所述的抗体与药物偶联体。
  10. 一种治疗或预防肿瘤的方法，其特征在于，所述方法包括步骤：给需要的对象施用权利要求1-6任一项所述的抗体与药物偶联体或权利要求7所述的药物组合物。
  11. 一种用于连接小分子药物和生物大分子的连接头L1，其特征在于，L1含有类似于vc(valine-citrulline，缬氨酸-瓜氨酸)的可以被蛋白酶B切断结构的ADC连接头，其中，瓜氨酸残基被赖氨酸残基所代替，且赖氨酸残基的活性氨基可以连接x个寡聚的聚乙二醇PEG。
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