WO2022218415A1 - 一种依喜替康类似物的结晶形式及其制备方法 - Google Patents

一种依喜替康类似物的结晶形式及其制备方法 Download PDF

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WO2022218415A1
WO2022218415A1 PCT/CN2022/087108 CN2022087108W WO2022218415A1 WO 2022218415 A1 WO2022218415 A1 WO 2022218415A1 CN 2022087108 W CN2022087108 W CN 2022087108W WO 2022218415 A1 WO2022218415 A1 WO 2022218415A1
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antibody
formula
crystal form
compound represented
ray powder
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PCT/CN2022/087108
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French (fr)
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吴琪
杨俊然
杜振兴
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江苏恒瑞医药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides

Definitions

  • the present disclosure relates to a crystalline form of an ixitecan analog and a preparation method thereof, in particular to a crystalline form of the compound represented by formula I and a preparation method.
  • Chemotherapy is still one of the most important anti-cancer methods including surgery, radiotherapy, and targeted therapy. Although there are many types of high-efficiency cytotoxins, the difference between tumor cells and normal cells is small, which limits the wide clinical application of these antitumor compounds due to their toxic and side effects. Anti-tumor monoclonal antibodies are specific to tumor cell surface antigens, and antibody drugs have become the front-line drugs for anti-tumor therapy. However, when antibodies are used alone as anti-tumor drugs, the efficacy is often unsatisfactory.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies binding to normal cells and tumor cell surface antigens and cytotoxicity, while avoiding the defects of low efficacy of the former and excessive toxic and side effects of the latter.
  • ADCs Antibody drug conjugates
  • the first antibody-drug conjugate Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals Co., Ltd.) was approved by the U.S. FDA for the treatment of acute myeloid leukemia (Drugs of the Future (Drugs of the Future). 2000) 25(7):686; US4970198; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).
  • Adcetris (brentuximab vedotin, Seattle Genetics) passed the US FDA fast track review for the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21 (7 ): 778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). It is a new type of targeted ADC drug, which can directly act on the target CD30 on lymphoma cells and undergo endocytosis to induce tumor cell apoptosis.
  • Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of HER2-positive patients with advanced or metastatic disease resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel breast cancer patients (WO2005037992; US8088387).
  • Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.
  • camptothecin derivatives which have antitumor effects by inhibiting topoisomerase I.
  • camptothecin derivative ixitecan chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H ,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione
  • ADC combined drugs
  • WO2020063676 relates to a series of new ligand-drug conjugates, among which a class of ligand-drug conjugates represented by the general formula (Pc-L-Y-Dr) has good antitumor activity, and its structure is as follows:
  • Pc represents a ligand
  • n is 1 to 10, which can be an integer or a decimal.
  • the compound shown in formula I can be used to prepare the above-mentioned ligand-drug conjugate, and the structure of the compound shown in formula I is as follows:
  • crystal structure of medicinal active ingredients and their intermediates often affects their chemical stability. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other forms. crystal form.
  • amorphous products do not have a regular crystal structure and often have other defects, such as poor product stability, finer crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, it is necessary to improve various properties of the above-mentioned products, and we need to conduct in-depth research to find new crystal forms with higher crystal purity and good chemical stability.
  • the present disclosure provides a new crystal form of the compound represented by formula I and a preparation method thereof.
  • the present disclosure provides a crystal form C of the compound represented by formula I, whose X-ray powder diffraction pattern has characteristic peaks at 2 ⁇ angles of 6.0, 8.8, 10.3, 12.2 and 15.5.
  • the present disclosure provides a crystalline form C of the compound of formula I, which has an X-ray powder diffraction pattern at 2 ⁇ angles of 6.0, 7.3, 8.8, 9.2, 10.3, 12.2, 15.5, 18.5 and There is a characteristic peak at 24.6.
  • the present disclosure provides a crystal form C of the compound of formula I, the X-ray powder diffraction pattern of which is shown in FIG. 4 .
  • the present disclosure provides a D crystal form of the compound represented by formula I, whose X-ray powder diffraction pattern at 2 ⁇ angles is 6.1, 7.3, 9.0, 10.6, 11.3, 11.5, 11.9, 12.5, 12.9, 14.6, 14.8, 15.9 , 16.1, 16.6, 17.4, 18.7, 19.4, 20.9, 21.6, 22.3, 23.1, 23.8, 24.9, 26.7, 28.3 and 29.8 have characteristic peaks.
  • the present disclosure provides a crystal form D of the compound of formula I, the X-ray powder diffraction pattern of which is shown in FIG. 5 .
  • the present disclosure provides a crystal form E of the compound represented by formula I, whose X-ray powder diffraction pattern at 2 ⁇ angles is 6.7, 7.1, 7.3, 7.6, 8.4, 10.2, 10.8, 11.9, 12.6, 13.4, 14.3, 15.2 , 15.6, 17.0, 17.9, 19.3, 20.4, 21.3 and 22.3 have characteristic peaks.
  • the present disclosure provides a crystalline form of the compound represented by formula I, the X-ray powder diffraction pattern of which is shown in FIG. 6 .
  • the present disclosure provides a crystal form F of the compound represented by formula I, whose X-ray powder diffraction pattern has characteristic peaks at 2 ⁇ angles of 9.5, 9.7, 11.2, 14.6, 14.8, 16.5, 17.2, 20.5, 22.7 and 28.6 .
  • the present disclosure provides a Form F of the compound of Formula I, the X-ray powder diffraction pattern of which is shown in FIG. 7 .
  • the present disclosure further provides a method for preparing crystal form C of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, and crystallization, and the solvent is tetrahydrofuran.
  • the present disclosure further provides a method for preparing the D crystal form of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, beating overnight for crystallization, and the solvent is acetonitrile.
  • the present disclosure further provides a method for preparing the E crystal form of the compound represented by the formula I, the method comprising: placing the A crystal form of the compound represented by the formula I under a humidity condition, and inverting the crystal.
  • the present disclosure further provides a method for preparing the F crystal form of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, beating for 12 hours for crystallization, and the solvent is acetonitrile.
  • the crystal form obtained in the present disclosure is subjected to structural determination and crystal form research.
  • crystallization methods of the crystal forms in the present disclosure are conventional, such as volatilization crystallization, cooling crystallization or crystallization at room temperature.
  • the starting material used in the preparation method of the crystal form of the present disclosure can be the compound represented by formula I in any form, and the specific form includes but is not limited to: amorphous, any crystal form, hydrate, solvate and the like.
  • the crystal form D in the present disclosure is prone to crystallize after drying, and the reproducibility of the crystal form E and F is poor.
  • the present disclosure also provides a preparation method of a ligand-drug conjugate represented by formula II or a pharmaceutically acceptable salt or solvate thereof, comprising: after reducing the ligand, mixing with formula I described in the present disclosure The steps of the crystal form coupling reaction of the compound shown,
  • Pc represents a ligand
  • n is 1 to 10, which can be an integer or a decimal.
  • the Pc is an antibody or an antigen-binding fragment thereof, the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti- HER4(ErbB4) Antibody, Anti-CD20 Antibody, Anti-CD22 Antibody, Anti-CD30 Antibody, Anti-CD33 Antibody, Anti-CD44 Antibody, Anti-CD56 Antibody, Anti-CD70 Antibody, Anti-CD73 Antibody, Anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin-C Antibody, Anti-SLC44A4 Antibody, Anti-CD20
  • the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab, or antigen-binding fragment thereof .
  • the "beating” described in the present disclosure refers to a method for purifying by utilizing the characteristics that substances have poor solubility in solvents but good solubility of impurities in solvents, and beating and purification can decolorize, change crystal forms or remove a small amount of impurities.
  • An "X-ray powder diffraction pattern or XRPD" as used in this disclosure is a pattern obtained by using Cu-K ⁇ radiation in an X-ray powder diffractometer.
  • Differential Scanning Calorimetry or DSC refers to measuring the temperature difference and heat flow difference between a sample and a reference during the heating or constant temperature of the sample to characterize all physical changes and chemical changes related to thermal effects. change to obtain the phase transition information of the sample.
  • the "2 ⁇ or 2 ⁇ angle" in the present disclosure refers to the diffraction angle, ⁇ is the Bragg angle, the unit is ° or degree, and the error range of 2 ⁇ is ⁇ 0.3 or ⁇ 0.2 or ⁇ 0.1.
  • interplanar spacing or interplanar spacing (d value) refers to the spatial lattice selection of three non-parallel unit vectors a, b, c connecting two adjacent lattice points.
  • the array is divided into juxtaposed parallelepiped units called interplanar spacing.
  • the space lattice is divided according to the determined parallelepiped unit connection, and a set of straight line grids is obtained, which is called space lattice or lattice.
  • Lattice and lattice use geometric points and lines to reflect the periodicity of the crystal structure. Different crystal planes have different interplanar spacings (that is, the distance between two adjacent parallel crystal planes); the unit is or Angstrom.
  • ligand-drug conjugate refers to a ligand linked to a biologically active drug through a stable linking unit.
  • the "ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to linking a monoclonal antibody or antibody fragment with a biologically active toxic drug through a stable linking unit .
  • ADC antibody-drug conjugate
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibodies described in the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, Anti-HER3 (ErbB3) Antibody, Anti-HER4 (ErbB4) Antibody, Anti-CD20 Antibody, Anti-CD22 Antibody, Anti-CD30 Antibody, Anti-CD33 Antibody, Anti-CD44 Antibody, Anti-CD56 Antibody, Anti-CD70 Antibody, Anti-CD73 Antibody, Anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin- One or more of
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine antibody in the present disclosure refers to the use of murine antibodies in accordance with the knowledge and skill in the art. In preparation, test subjects are injected with a specific antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To build a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the mouse variable region gene from the mouse hybridoma cell. After connecting with human constant region gene into chimeric gene, it is inserted into expression vector, and finally chimeric antibody molecule is expressed in eukaryotic system or prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody antibodies produced in framework sequences.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al.
  • humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of the CDRs by phage display. Documents further describing methods involved in humanization using mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the technologies related to the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising A bivalent fragment of two Fab fragments linked by a disulfide bridge, (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv fragment composed of the one-armed VH and VL domains of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs)
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at the 224th position of the H chain), wherein the N-terminal side of the H chain is an antibody fragment. About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody with a molecular weight of about 100,000 obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG with the enzyme pepsin and having antigen-binding activity and comprising two Fab regions linked at the hinge position Fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment of the antibody can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.
  • single chain antibody is intended to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker molecule.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof, eg using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to the heavy chain variable domain CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-7 M, eg, about less than 10-8 M , 10-9 M, or 10-10 M or less.
  • KD affinity
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.
  • a heterocyclic group optionally substituted with an alkyl group means that an alkyl group may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group .
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • pharmaceutically acceptable salt refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, when such salts are used in mammals With safety and efficacy, and with due biological activity, the antibody-antibody drug conjugate compound of the present disclosure contains at least one amino group, so it can form salts with acids.
  • Non-limiting examples of pharmaceutically acceptable salts include: salts acid salt, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, Dihydrogen phosphate, salicylates, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonic acid salt, p-toluenesulfonate.
  • solvate refers to a ligand-drug conjugate compound of the present disclosure that forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile , isopropanol, DMSO, ethyl acetate.
  • drug loading refers to the average amount of cytotoxic drug loaded on each ligand in the molecule of formula (I), and can also be expressed as the ratio of the amount of drug to the amount of antibody, and the drug loading can range from each ligand. (Pc) 0-12, preferably 1-10 cytotoxic drugs (D) are linked.
  • the drug loading is represented as n, and an exemplary value may be the mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the average amount of drug product per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay and HPLC.
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through the linking unit.
  • the cytotoxic drug can be coupled to the antibody in the coupling reaction.
  • the number of drug molecules will be less than the theoretical maximum.
  • Ligand cytotoxic drug conjugate loading can be controlled by the following non-limiting methods, including:
  • carrier is used for the drugs of the present disclosure, and refers to a system capable of changing the way the drug enters the human body and its distribution in the body, controlling the release rate of the drug, and delivering the drug to the targeted organs.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • Figure 1 is an amorphous XRPD pattern of the compound of formula I.
  • Figure 2 is the XRPD pattern of the A crystal form of the compound represented by formula I.
  • Figure 3 is the XRPD pattern of the B crystal form of the compound represented by formula I.
  • Figure 4 is the XRPD pattern of the C crystal form of the compound represented by formula I.
  • Figure 5 is the XRPD pattern of the D crystal form of the compound represented by formula I.
  • Figure 6 is the XRPD pattern of the E crystal form of the compound represented by formula I.
  • Figure 7 is the XRPD pattern of the F crystal form of the compound represented by formula I.
  • Cu-K ⁇ 1 wavelength is The wavelength of Cu-K ⁇ 2 is The Cu-K ⁇ wavelength is the weighted average of K ⁇ 1 and K ⁇ 2 )
  • the compound represented by formula I was prepared according to Example 9 of WO2020063676, the obtained product was detected as amorphous by X-ray powder diffraction, and the XRPD spectrum was shown in FIG. 1 .
  • Example 4 150 mg of the compound represented by formula I was added to 4 mL of tetrahydrofuran, 1 mg of seed crystal (Example 4) was added, stirred at room temperature, centrifuged, and dried in vacuo to obtain the product. Through X-ray powder diffraction detection, the product is C crystal form.
  • the target product was obtained by placing 10 mg of Form A under 75% RH and 93% RH humidity conditions for 7 days or more.
  • the product is defined as E crystal form
  • the XRPD spectrum is shown in Figure 6, and the characteristic peak positions are shown in Table 5.

Abstract

一种依喜替康类似物的结晶形式及其制备方法，具体而言，为一种式I所示化合物的结晶形式及其制备方法。所述晶型具备良好的理化性质，更有利于原料的存储与利用。

Description

一种依喜替康类似物的结晶形式及其制备方法
本申请要求申请日为2021年4月15日的中国专利申请2021104069487的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开涉及一种依喜替康类似物的结晶形式及其制备方法，具体地涉及式I所示化合物的结晶形式及制备方法。
背景技术
化疗依然是包括手术、放疗、以及靶向治疗法在内的最重要的抗癌手段之一。尽管高效细胞毒素的种类很多，但是肿瘤细胞和正常细胞之间差别很小，限制了这些抗肿瘤化合物由于毒副作用在临床上的广泛应用。而抗肿瘤单克隆抗体对于肿瘤细胞表面抗原的特异性，抗体药物已成为抗肿瘤治疗的前线药物，但单独使用抗体作为抗肿瘤药物时，疗效经常不尽人意。
抗体药物偶联物(antibody drug conjugate，ADC)将单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒素的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低对正常细胞的影响(Mullard A,(2013)Nature Reviews Drug Discovery,12:329–332；DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814)。
2000年第一个抗体药物偶联物Mylotarg(吉妥珠单抗奥唑米星(gemtuzumab ozogamicin)，惠氏制药有限公司)被美国FDA批准上市，用于治疗急性髓细胞白血病(Drugs of the Future(2000)25(7):686；US4970198；US 5079233；US 5585089；US 5606040；US 5693762；US 5739116；US 5767285；US 5773001)。
2011年8月，Adcetris(brentuximab vedotin，西雅图基因遗传公司)通过美国FDA快速审评通道，用于治疗霍奇金淋巴瘤以及复发性间变性大细胞淋巴瘤(Nat.Biotechnol(2003)21(7):778-784；WO2004010957；WO2005001038；US7090843A；US7659241；WO2008025020)。
Figure PCTCN2022087108-appb-000001
是一种新型靶向ADC药物，能使药物直接作用于淋巴瘤细胞上的靶点CD30后发生内吞作用从而诱导肿瘤细胞的凋亡。
Mylotarg和Adcetris都是针对血液肿瘤进行靶向治疗，血液肿瘤和实体肿瘤相比组织结构相对简单。2013年2月，Kadcyla(ado-trastuzumab emtansine，T-DM1)获得美国FDA批准，用于治疗HER2阳性同时对曲妥珠单抗(Tratuzumab，商品名：Herceptin)和紫杉醇有抗药性的晚期或转移性乳腺癌患者(WO2005037992；US8088387)。Kadcyla 是美国FDA批准的治疗实体肿瘤的第一个ADC药物。
用于抗体药物偶联物的具有细胞毒性的小分子有几类，其中有一类是喜树碱衍生物，它们通过抑制拓扑异构酶I而具有抗肿瘤作用。报道喜树碱衍生物依喜替康(化学名：(1S,9S)-1-氨基-9-乙基-5-氟-2,3-二氢-9-羟基-4-甲基-1H,12H-苯并[de]吡喃并[3’,4’:6,7]咪唑并[1,2-b]喹啉-10,13(9H,15H)-二酮)应用于抗体偶联药物(ADC)的文献有WO2014057687；Clinical Cancer Research(2016)22(20)：5097-5108；Cancer Sci(2016)107:1039-1046。
WO2020063676涉及一系列新的配体-药物偶联物，其中一类通式为(Pc-L-Y-Dr)所示的配体-药物偶联物具有良好的抗肿瘤活性，其结构如下所示：
Figure PCTCN2022087108-appb-000002
其中，Pc表示配体，n为1至10，可以为整数，也可以为小数。
另外，式I所示化合物可用于制备上述配体-药物偶联物，式I所示化合物的结构如下所示：
Figure PCTCN2022087108-appb-000003
药用的活性成分及其中间体的晶型结构往往影响到该他们的化学稳定性，结晶条件及储存条件的不同有可能导致化合物的晶型结构的变化，有时还会伴随着产生其他形态的晶型。一般来说，无定型的产品没有规则的晶型结构，往往具有其它缺陷，比如产物稳定性较差，析晶较细，过滤较难，易结块，流动性差等。因此，改善上述产物的各方面性质是很有必要的，我们需要深入研究找到晶型纯度较高并且具备良好化学稳定的新晶型。
发明内容
本公开提供了一种式I所示化合物新的晶型及其制备方法。
Figure PCTCN2022087108-appb-000004
本公开提供了一种式I所示化合物的C晶型，其X-射线粉末衍射图谱在2θ角为6.0、8.8、10.3、12.2和15.5处有特征峰。
在某些的实施方案中，本公开提供一种式I所示化合物的C晶型，其X-射线粉末衍射图谱在2θ角为6.0、7.3、8.8、9.2、10.3、12.2、15.5、18.5和24.6处有特征峰。
在某些的实施方案中，本公开提供一种式I所示化合物的C晶型，其X-射线粉末衍射图谱如图4所示。
本公开提供了一种式I所示化合物的D晶型，其X-射线粉末衍射图谱在2θ角为6.1、7.3、9.0、10.6、11.3、11.5、11.9、12.5、12.9、14.6、14.8、15.9、16.1、16.6、17.4、18.7、19.4、20.9、21.6、22.3、23.1、23.8、24.9、26.7、28.3和29.8处有特征峰。
在某些的实施方案中，本公开提供一种式I所示化合物的D晶型，其X-射线粉末衍射图谱如图5所示。
本公开提供了一种式I所示化合物的E晶型，其X-射线粉末衍射图谱在2θ角为6.7、7.1、7.3、7.6、8.4、10.2、10.8、11.9、12.6、13.4、14.3、15.2、15.6、17.0、17.9、19.3、20.4、21.3和22.3处有特征峰。
在某些的实施方案中，本公开提供一种式I所示化合物的E晶型，其X-射线粉末衍射图谱如图6所示。
本公开提供了一种式I所示化合物的F晶型，其X-射线粉末衍射图谱在2θ角为9.5、9.7、11.2、14.6、14.8、16.5、17.2、20.5、22.7和28.6处有特征峰。
在某些的实施方案中，本公开提供一种式I所示化合物的F晶型，其X-射线粉末衍射图谱如图7所示。
本公开进一步提供一种制备式I所示化合物的C晶型的方法，所述方法包括：将式I所示化合物与适量的溶剂混合，结晶析出，所述溶剂为四氢呋喃。
本公开进一步提供一种制备式I所示化合物的D晶型的方法，所述方法包括：将式I所示化合物与适量的溶剂混合，打浆过夜析晶，所述溶剂为乙腈。
本公开进一步提供一种制备式I所示化合物的E晶型的方法，所述方法包括：将式I所示化合物的A晶型放置于湿度条件下，转晶。
本公开进一步提供一种制备式I所示化合物的F晶型的方法，所述方法包括：将式I所示化合物与适量的溶剂混合，打浆12h析晶，所述溶剂为乙腈。
通过X-射线粉末衍射图谱(XRPD)、差示扫描量热分析(DSC)对本公开所得到晶型进 行结构测定、晶型研究。
本公开中晶型的析晶方法是常规的，例如挥发析晶、降温析晶或室温下析晶。
本公开晶型制备方法中所用的起始原料可以是任意形式的式I所示化合物，具体形式包括但不限于：无定形、任意晶型、水合物、溶剂合物等。
本公开中的D晶型在干燥后容易发生转晶，E晶型和F晶型的重现性较差。
本公开还提供了一种式II所示的配体-药物偶联物或其药学上可接受的盐或溶剂合物的制备方法，包括：还原配体后，与本公开所述的式I所示化合物的晶型偶联反应的步骤，
Figure PCTCN2022087108-appb-000005
其中，Pc表示配体，n为1至10，可以为整数，也可以为小数。
在某些实施方式中，所述Pc为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体或全人源抗体；优选为单克隆抗体。
在某些实施方式中，其中所述的抗体或其抗原结合片段选自抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体或抗Mesothelin抗体或其抗原结合片段。
在某些实施方式中，其中所述的抗体或其抗原结合片段选自Trastuzumab、Pertuzumab、Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab，或其抗原结合片段。
在本申请的说明书和权利要求书中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。然而，为了更好地理解本公开，下面提供了部分相关术语的定义和解释。另外，当本申请所提供的术语的定义和解释与本领域技术人员所通常理解的含义不一致时，以本申请所提供的术语的定义和解释为准。
本公开所述的“打浆”是指利用物质在溶剂中溶解性差，但杂质在溶剂中溶解性好的 特性进行纯化的方法，打浆提纯可以去色、改变晶型或去除少量杂质。
本公开所述的“X-射线粉末衍射图谱或XRPD”是指根据布拉格公式2d sinθ＝nλ(式中，λ为X射线的波长，衍射的级数n为任何正整数，一般取一级衍射峰，n＝1)，当X射线以掠角θ(入射角的余角，又称为布拉格角)入射到晶体或部分晶体样品的某一具有d点阵平面间距的原子面上时，就能满足布拉格方程，从而测得了这组X射线粉末衍射图。
本公开所述的“X-射线粉末衍射图谱或XRPD”是通过在X-射线粉末衍射仪中使用Cu-Kα辐射得到的图谱。
本公开所述的“差示扫描量热分析或DSC”是指在样品升温或恒温过程中，测量样品与参考物之间的温度差、热流差，以表征所有与热效应有关的物理变化和化学变化，得到样品的相变信息。
本公开所述的“2θ或2θ角度”是指衍射角，θ为布拉格角，单位为°或度，2θ的误差范围为±0.3或±0.2或±0.1。
本公开所述的“晶面间距或晶面间距(d值)”是指空间点阵选择3个不相平行的连结相邻两个点阵点的单位矢量a，b，c，它们将点阵划分成并置的平行六面体单位，称为晶面间距。空间点阵按照确定的平行六面体单位连线划分，获得一套直线网格，称为空间格子或晶格。点阵和晶格是分别用几何的点和线反映晶体结构的周期性，不同的晶面，其面间距(即相邻的两个平行晶面之间的距离)各不相同；单位为
Figure PCTCN2022087108-appb-000006
或埃。
术语“配体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本公开所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体或抗 Mesothelin抗体中一个或多个；优选为曲妥珠单抗(Trastuzumab，商品名Herceptin)、帕妥珠单抗(Pertuzumab，也被称作2C4，商品名Perjeta)、尼妥珠单抗(Nimotuzumab，商品名泰欣生)、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其 抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人 (1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 - 9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开配体-药物偶联物的盐，或本公开中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本公开抗体-抗体药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂化物”或“溶剂化合物”指本公开的配体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”是指式(I)分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接0-12个，优 选1-10个细胞毒性药物(D)。在本公开的实施方式中，载药量表示为n，示例性的可以为1，2，3，4，5，6，7，8，9，10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本公开的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
附图说明
图1为式I所示化合物的无定形的XRPD图谱。
图2为式I所示化合物的A晶型的XRPD图谱。
图3为式I所示化合物的B晶型的XRPD图谱。
图4为式I所示化合物的C晶型的XRPD图谱。
图5为式I所示化合物的D晶型的XRPD图谱。
图6为式I所示化合物的E晶型的XRPD图谱。
图7为式I所示化合物的F晶型的XRPD图谱。
具体实施方式
以下将结合实施例更详细地解释本公开，本公开的实施例仅用于说明本公开的技术方案，并非限定本公开的实质和范围。
试验所用仪器的测试条件：
1、差示扫描量热仪(Differential Scanning Calorimeter,DSC)
仪器型号：Mettler Toledo DSC 3+
吹扫气：氮气
升温速率：10.0℃/min
温度范围：25-300℃(25-200℃)
2、X-射线衍射谱(X-ray Powder Diffraction,XRPD)
仪器型号：BRUKER D8 ADVANCE X-射线粉末衍射仪
射线：单色Cu-Kα射线(Cu-Kα1波长为
Figure PCTCN2022087108-appb-000007
Cu-Kα2波长为
Figure PCTCN2022087108-appb-000008
Cu-Kα波长取Kα1与Kα2的加权平均值
Figure PCTCN2022087108-appb-000009
)
扫描方式：θ/2θ，扫描范围：3-45°，
电压：40KV，电流：40mA；
实施例1
根据WO2020063676的实施例9制备式I所示化合物，得到的产物经X-射线粉末衍射检测为无定形，XRPD谱图如图1所示。
实施例2
将10mg式I所示化合物加入1mL二氯甲烷，室温搅拌，离心，真空干燥后得到产物。经X-射线粉末衍射检测，将该产物定义为A晶型，XRPD谱图如图2所示，其特征峰位置如表1所示。DSC结果显示，吸热峰为141.78℃、162.11℃
表1
Figure PCTCN2022087108-appb-000010
实施例3
将10mg式I所示化合物加入0.4mL乙腈，室温搅拌，离心，真空干燥后得到产物。经X-射线粉末衍射检测，将该产物定义为B晶型，XRPD谱图如图3所示，其特征峰位置如表2所示。DSC结果显示，吸热峰为142.10℃。
表2
Figure PCTCN2022087108-appb-000011
实施例4
将10mg式I所示化合物加入1mL四氢呋喃，室温搅拌，离心，真空干燥后得到产物。经X-射线粉末衍射检测，将该产物定义为C晶型，XRPD谱图如图4，其特征峰位置如表3所示。DSC结果显示，吸热峰为153.85℃。
表3
Figure PCTCN2022087108-appb-000012
Figure PCTCN2022087108-appb-000013
实施例5
将10mg式I所示化合物加入1mL四氢呋喃，加热至50℃溶清，降至室温搅拌，离心，真空干燥后得到产物。经X-射线粉末衍射检测，该产物为C晶型。
实施例6
将150mg式I所示化合物加入4mL四氢呋喃，加入1mg晶种(实施例4)，室温搅拌，离心，真空干燥后得到产物。经X-射线粉末衍射检测，该产物为C晶型。
实施例7
将150mg式I所示化合物加入3mL乙腈，室温搅拌过夜，离心，得到产物。经X-射线粉末衍射检测，将该产物定义为D晶型，XRPD谱图如图5，其特征峰位置如表4所示。
表4
Figure PCTCN2022087108-appb-000014
Figure PCTCN2022087108-appb-000015
实施例8
将10mg晶型A置于75％RH和93％RH湿度条件下7天及以上，得到目标产物。经X-射线粉末衍射检测，将该产物定义为E晶型，XRPD谱图如图6，其特征峰位置如表5所示。
表5
Figure PCTCN2022087108-appb-000016
Figure PCTCN2022087108-appb-000017
实施例9
将15mg晶型A置于动态水分吸附仪中，经过引湿性实验(湿度梯度：50％-95％-0％-95％-0％-50％RH)，得到目标产物。经X-射线粉末衍射检测，该产物为E晶型。
实施例10
将150mg式I所示化合物加入4mL乙腈，室温搅拌12小时，离心，真空干燥后得到产物。经X-射线粉末衍射检测，将该产物定义为F晶型，XRPD谱图如图7，其特征峰位置如表6所示。
表6
Figure PCTCN2022087108-appb-000018
实施例11
将A、B、C晶型样品敞口平摊放置，分别考察高温(40℃、60℃)和高湿(RH 75％、RH 92.5％)条件下样品的稳定性，取样考察期为30天，结果见表7。
表7
Figure PCTCN2022087108-appb-000019
实施例11
将C晶型样品分别放置于-20℃，4℃，25℃/60％RH和40℃/75％RH条件下考察其稳定性，结果见表8。
表8
Figure PCTCN2022087108-appb-000020

Claims (13)

  1. 一种式I所示化合物的C晶型，其X-射线粉末衍射图谱在2θ角为6.0、8.8、10.3、12.2和15.5处有特征峰，
    Figure PCTCN2022087108-appb-100001
  2. 根据权利要求1所述的式I所示化合物的C晶型，其X-射线粉末衍射图谱在2θ角为6.0、7.3、8.8、9.2、10.3、12.2、15.5、18.5和24.6处有特征峰。
  3. 根据权利要求1所述的式I所示化合物的C晶型，其X-射线粉末衍射图谱如图4所示。
  4. 一种式I所示化合物的D晶型，其X-射线粉末衍射图谱在2θ角为6.1、7.3、9.0、10.6、11.3、11.5、11.9、12.5、12.9、14.6、14.8、15.9、16.1、16.6、17.4、18.7、19.4、20.9、21.6、22.3、23.1、23.8、24.9、26.7、28.3和29.8处有特征峰。
  5. 根据权利要求4所述的式I所示化合物的D晶型，其X-射线粉末衍射图谱如图5所示。
  6. 一种式I所示化合物的E晶型，其X-射线粉末衍射图谱在2θ角为6.7、7.1、7.3、7.6、8.4、10.2、10.8、11.9、12.6、13.4、14.3、15.2、15.6、17.0、17.9、19.3、20.4、21.3和22.3处有特征峰。
  7. 根据权利要求6所述的式I所示化合物的E晶型，其X-射线粉末衍射图谱如图6所示。
  8. 一种式I所示化合物的F晶型，其X-射线粉末衍射图谱在2θ角为9.5、9.7、11.2、14.6、14.8、16.5、17.2、20.5、22.7和28.6处有特征峰。
  9. 根据权利要求8所述的式I所示化合物的F晶型，其X-射线粉末衍射图谱如图7 所示。
  10. 根据权利要求1-9任意一项所述的式I所示化合物的晶型，其中所述2θ角的误差范围为±0.2。
  11. 一种制备如权利要求1-3任意一项所述的式I所示化合物的C晶型的方法，所述方法包括：将式I所示化合物与适量的溶剂混合，结晶析出，所述溶剂为四氢呋喃。
  12. 一种式II所示的配体-药物偶联物或其药学上可接受的盐或溶剂合物的制备方法，包括还原配体后，与所述的式I所示化合物的晶型偶联反应的步骤，
    Figure PCTCN2022087108-appb-100002
    其中，Pc表示配体，n为1至10，可以为整数，也可以为小数。
  13. 根据权利要求12所述的制备方法，所述Pc为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体或全人源抗体，优选为单克隆抗体，更优选HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体或抗Mesothelin抗体或其抗原结合片段。
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WO2020063673A1 (zh) * 2018-09-30 2020-04-02 江苏恒瑞医药股份有限公司 抗b7h3抗体-依喜替康类似物偶联物及其医药用途
WO2020063676A1 (zh) * 2018-09-26 2020-04-02 江苏恒瑞医药股份有限公司 依喜替康类似物的配体-药物偶联物及其制备方法和应用
CN111051330A (zh) * 2017-08-31 2020-04-21 第一三共株式会社 抗体-药物缀合物的改进制备方法
CN111689980A (zh) * 2019-05-26 2020-09-22 四川百利药业有限责任公司 一种喜树碱药物及其抗体偶联物

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CN111051330A (zh) * 2017-08-31 2020-04-21 第一三共株式会社 抗体-药物缀合物的改进制备方法
WO2020063676A1 (zh) * 2018-09-26 2020-04-02 江苏恒瑞医药股份有限公司 依喜替康类似物的配体-药物偶联物及其制备方法和应用
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