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CN-115279416-B - Reactive conjugates

CN115279416BCN 115279416 BCN115279416 BCN 115279416BCN-115279416-B

Abstract

The present invention relates to compounds (reactive conjugates) for chemically modifying therapeutic antibodies or proteins. These compounds are capable of selectively attaching a payload region to an antibody or antibody fragment in a single step, thereby producing a modified antibody or modified antibody fragment that can be used to diagnose, monitor, image, or treat a disease.

Inventors

  • Origen Franz Nyangal
  • Jean Manuel Segura
  • PATRICK GARRETT
  • Victoria Bostopalenko
  • LEO MARX
  • Frederick Levy

Assignees

  • 德彪药业国际股份公司

Dates

Publication Date
20260508
Application Date
20201203
Priority Date
20191203

Claims (20)

  1. 1. A compound represented by the following formula (1): P-Y-S-V (1) Wherein, the P is the payload; Y is a reactive moiety capable of reacting with a side chain of an amino acid; V is a vector capable of interacting with the crystallizable fragment Fc region of an antibody or antibody fragment; S is a spacer having a length Z, wherein Z is a length such that when the vector V interacts with the crystallizable fragment Fc region of the antibody or antibody fragment, the reactive moiety Y is capable of reacting with a side chain of an amino acid residue on the antibody or antibody fragment; wherein the reactive moiety is represented by one of the following formulas: (6a) (6b) (6c) (6d) (6e) (6j) (6l) (6m) (6n) (6t) (6v) (6y) (6a’) (6j’) (6k’) (6l’) Wherein, the Refers to covalent attachment to spacer S, and Refers to covalent attachment to payload P, and Wherein the spacer is (A2) A group represented by the following formula (7): –X 1 –(CH 2 CH 2 O) n2 –CH 2 CH 2 –X 2 –(7) Wherein, the X 1 is NH, O, or S; X 2 is NH or C=O, and N2 is an integer from 6 to 28, or (B2) A peptide group having 6 to 25 amino acids in the main chain, each amino acid being selected from the group consisting of Pro, gly and Ser; the carrier is a peptide represented by one of the following formulas (8 a) and (8 b): Axx is Asp; bxx is Trp; Cxx is His; dxx is Leu; exx is Gly; fxx is Glu; Gxx is Thr; Hxx represents a single covalent bond or a trifunctional amino acid selected from the group consisting of 2, 3-diamino-propionic acid Dap, 2, 4-diamino-butyric acid Dab, lys, guanylic acid Orn and homolysine; Z 1 represents If Hxx is a single covalent bond, Z 1 represents a group covalently bound to the C-terminal end of Gxx, said group being selected from the group consisting of-N (H) (R), wherein R represents a hydrogen atom, an alkyl or cycloalkyl group, or a moiety of a compound containing a binding group selected from the group consisting of biotin, dibenzocyclooctyne DBCO, trans-cyclooctene TCO, bicyclo [6.1.0] nonene BCN, alkyne, azide, bromoacetamide, maleimide and thiol; if Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, Z 1 represents a group covalently bound to the C-terminal end of Hxx, said group being selected from the group consisting of-N (H) (R) wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group, or If Hxx is a trifunctional amino acid and Y' is bound to the C-terminus of Hxx, Z 1 represents a hydrogen atom bound to the side chain of Hxx; Z 2 represents If Hxx is a single covalent bond, Z 2 represents a group covalently bound to the N-terminus of Axx, selected from a hydrogen atom, a carbonyl-containing group, or a moiety of a compound containing a binding group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide, and thiol; If Hxx is a trifunctional amino acid and Y' is bound to the side chain of Hxx, Z 2 represents a group covalently bound to the N-terminus of Hxx, selected from a hydrogen atom or a carbonyl-containing group, or If Hxx is a trifunctional amino acid and Y' is bound to the N-terminus of Hxx, Z 2 represents a hydrogen atom bound to the side chain of Hxx; if Hxx is a trifunctional amino acid, only Y' is present, and If Z 1 is bound to the C-terminus of Hxx, or if Z 2 is bound to the N-terminus of Hxx, Y' represents a moiety covalently bound to the side chain of Hxx, If Z 1 binds to the side chain of Hxx, Y' represents a moiety covalently bound to the C-terminus of Hxx, or If Z 2 is bound to the side chain of Hxx, Y' represents a moiety covalently bound to the N-terminus of Hxx; y' is selected from compounds containing a binding group selected from biotin, DBCO, TCO, BCN, alkyne, azide, bromoacetamide, maleimide or thiol; x 3 represents a single covalent bond or a divalent group comprising one or more atoms selected from carbon, nitrogen and oxygen; Refers to covalent attachment to the spacer S.
  2. 2. The compound of claim 1, wherein the antibody fragment is incorporated into an Fc-fusion protein.
  3. 3. The compound of claim 1 or 2, wherein the payload comprises one selected from the group consisting of: (i) a labeling moiety comprising a radionuclide, Chromophore, or A fluorophore; (ii) A moiety comprising a conjugated diene, tetrazine TZ, alkyne or azide, DBCO, TCO, BCN, or (Iii) Selected from the group consisting of: An antitumor agent; RNA-polymerase II inhibitors; Antimetabolites; A kinase inhibitor; Immunomodulators, or An anti-infective agent.
  4. 4. The compound of claim 3, wherein the anti-neoplastic agent comprises a topoisomerase inhibitor, a DNA-cleaving agent, or an anti-mitotic agent.
  5. 5. The compound of claim 1 or 2, wherein the payload is a chelator.
  6. 6. The compound of claim 5, wherein the chelator chelates radionuclides.
  7. 7.A compound according to claim 5 wherein the chelating agent is selected from the group consisting of diethylenetriamine pentaacetic acid DTPA, cyclohexyldiethylenetriamine pentaacetic acid CH-X-DTPA, deferoxamine DFO, 1- (1, 3-carboxypropyl) -4, 7-carboxymethyl-1, 4, 7-tetraacetic acid NODAGA, 1,4,7, 10-tetraazacyclododecane-1-glutaric acid-4, 7, 10-triacetic acid DOTAGA, 2' - (1, 4, 7-triazacyclononane-1, 4-diyl) diacetate NO2A, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid DOTA, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid NOTA, ethylenediamine tetraacetic acid EDTA, ethylenediamine diacetic acid, triethylenetetramine hexaacetic acid TTHA, 1,4,8, 11-tetraazacyclotetradecane CYCLAM, 1,4,8, 11-tetraazacyclotetradecane-1, 4,8, 11-tetraacetic acid TETA, 1,4,8, 11-tetraazabicyclo [6.6.2] hexadecane-4, 11-diacetic acid CB-TE2A, 2'' - (1, 4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetamide 3AM, 1,4,7, 10-tetraazacyclododecane-1, 7-diacetic acid DO2A, 1,5, 9-triazacyclododecane TACD, (3 a1s,5a1 s) -decahydro-3 a,5a,8a,10 a-tetraazapyrene, 1,4, 7-triazacyclononane TACN, 1,4,7, 10-tetraazacyclododecane, tris (hydroxypyridinone) THP, 3- (((4, 7-bis ((hydroxy (hydroxymethyl) phosphoryl) methyl) -1,4, 7-triazolin-1-yl) methyl) (hydroxy) phosphoryl) propionic acid NOPO, 3,6,9,15-tetraazabicyclo [9.3.1] pentadecane-1 (15), 11, 13-triene-3, 6, 9-triacetic acid PCTA, 2,2' ',2' ' ' - (1, 4,7, 10-tetraazatridecyl-1, 4,7, 10-tetrayl) tetraacetic acid TRITA, 2' ',2' ' ' - (1, 4,7, 10-tetraazatridecyl-1, 4,7, 10-tetrayl) tetraacetamide TRITAM, 2',2' ' - (1, 4,7, 10-tetraazacyclotridec-1, 4, 7-triyl) triacetamide TRITRAM, trans-N-dimethylcyclopropylamine, 2' ' - (1, 4, 7-triazacyclononane-1, 4, 7-triyl) triacetamide NOTAM, Oxycyclolamide, dioxa-4, 10-diazacyclododecane, crosslinked bridged cyclolamide, triazacyclononane phosphonite TRAP, bispyridoxal diphosphate DPDP, meso-tetrakis (4-sulfonylphenyl) porphine TPPS 4 , ethylenebishydroxyphenylglycine EHPG, hexamethylenediamine tetraacetic acid, dimethylphosphinomethane DMPE, methylenediphosphonic acid, or dimercaptosuccinic acid DMPA.
  8. 8. The compound of claim 6, wherein the radionuclide is selected from the group ： 124 I、 131 I、 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb、 212 Pb、 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、 223 Ra、 97 Ru、 149 Tb、 152 Tb、 161 Tb、 99m Tc、 226 Th、 227 Th、 201 Tl、 89 Sr、 44 Sc、 43 Sc、 47 Sc、 153 Sm、 133 Xe or Al 18 F consisting of.
  9. 9. The compound of claim 7, wherein the chelator chelates a radionuclide, and wherein the radionuclide is selected from the group consisting of ： 124 I、 131 I、 86 Y、 90 Y、 177 Lu、 111 In、 188 Re、 55 Co、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 203 Pb、 212 Pb、 212 Bi、 213 Bi、 72 As、 211 At、 225 Ac、 223 Ra、 97 Ru、 149 Tb、 152 Tb、 161 Tb、 99m Tc、 226 Th、 227 Th、 201 Tl、 89 Sr、 44 Sc、 43 Sc、 47 Sc、 153 Sm、 133 Xe or Al 18 F.
  10. 10. The compound of claim 1 or 2, wherein the payload is selected from the group consisting of irinotecan, PNU-159582, amanitine, duocarmycin, auristatin, maytansine, tubulysin, kar Li Jimei, SN-38, paclitaxel, daunomycin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine, a pyrrole spindle kinesin KSP inhibitor, or indoline-benzodiazepine dimer.
  11. 11. The compound according to claim 1 or 2, wherein P is represented by the following formula (2): P 1 -L-- (2) Wherein, the P 1 is a payload as defined in any one of claims 3 to 10; L is a linker; Refers to covalent attachment to the reactive moiety Y.
  12. 12. The compound of claim 11, wherein the linker is one of: (a1) An alkylene group having 1 to 12 carbon atoms; (b1) A polyalkylene oxide group having 2 or 3 carbon atoms and having 1 to 36 repeating units, or (C1) A peptide group having 2 to 12 amino acids.
  13. 13. The compound of claim 1 or 2, which is one of the following: 、 、 、 、 、 、 、 Or alternatively, the first and second heat exchangers may be, ; Wherein, the P is as defined in any one of claims 3 to 10, and Y' is as defined in claim 1.
  14. 14. The compound of claim 1 or 2, which is one of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Or alternatively, the first and second heat exchangers may be, 。
  15. 15. A kit for site-specific modification of an antibody or antibody fragment, the kit comprising a compound according to any one of claims 1 to 14 and a buffer.
  16. 16. The kit of claim 15 for use in the regioselective modification of an antibody or antibody fragment, wherein the compound is immobilized on a solid phase substrate.
  17. 17. A method for regioselectively modifying an antibody or antibody fragment, the method comprising reacting an antibody or fragment thereof with a compound according to any one of claims 1 to 14.
  18. 18. The method of claim 17, wherein, The antibody is monoclonal antibody, or The antibody fragment is incorporated into an Fc-fusion protein.
  19. 19. The method of claim 18, wherein The antibody is adalimumab, a Du Nashan antibody, an alemtuzumab, an avermectin antibody, a bapidem antibody, a basiliximab, a Bei Tuo momab, a Bei Maiji antibody, a besiezomib antibody, a bevacizumab, a Bei Luotuo Shu Shan antibody, a native toxib antibody, a vitamin b toxib antibody, a buddamab antibody, a bonafida antibody, a katuxedan antibody, a cimetidine antibody, a cetuximab antibody, a Xin Panai antibody, a Krituximab, tetan-Krituximab, daclizumab, denomumab, denotuximab, dewar Lu Shankang, edentum monoclonal antibody, and pharmaceutical composition erlotinib, epratuzumab, enfratuzumab, vitamin-enfratuzumab, epratuzumab, ada-group mab, gemtuzumab, ji Tuo-ximab, golnemab, temozolomab, inbizumab, infliximab, oxuzumab, ipilimumab, ai Satuo, ipilimumab, j591 PSMA-antibody, la Bei Zhushan, lycra mab, mo Geli, bevacizumab Nexituzumab, nituzumab, natalizumab, nawuzumab, origani mab, ofamazumab, olamab, orgo Fu Shan, panitumumab, pembrolizumab Nexituzumab, nituzumab, natalizumab, nawuzumab, origani-Begoniab ofatuzumab, olauzumab, ago3592, panitumumab, pembrolizumab, zeigunder monoclonal antibody Zaleukinumab, zaleukinumab zeiginemab, zafimbriata mab, zaleukinumab Zalemtuzumab or fragments thereof; or alternatively The antibody fragment is incorporated into an Fc-fusion protein that is berazepine, aflibercept, ziv-aflibercept, duloxetine, risperidone, romidepsin, abamectin, or albazepine.
  20. 20. A modified antibody or modified antibody fragment obtained by reacting an antibody or antibody fragment with a compound according to any one of claims 1 to 14.

Description

Reactive conjugates Technical Field The present invention relates to chemically modified compounds (hereinafter sometimes referred to as "reactive conjugates") for therapeutic antibodies. These compounds are capable of selectively attaching a payload region to an antibody or antibody fragment in a single step, thereby producing a modified antibody or modified antibody fragment that can be used to diagnose, monitor, image, or treat a disease. Background Traditional cancer treatments (e.g., chemotherapy) are not only very laborious (because of their toxicity causing serious side effects), but can also be very occasional, with treatments effective on one patient, but not on the other. Thus, there is an ongoing need to develop new less toxic and/or more effective therapies, as is the ability to monitor the efficacy of the therapy, e.g., to be able to distinguish between "responders" and "non-responders" patients. To respond to these demands, a novel therapeutic agent called an Antibody-drug-conjugates (ADC) has emerged. ADCs utilize the targeting ability of antibodies (e.g., monoclonal antibodies (Monoclonal antibodies, mabs)) to deliver payloads (e.g., cytotoxic or labeling agents) directly to cancer cells. Targeting of cancer cells maximizes the therapeutic effect of the payload while minimizing toxic effects on healthy cells. Depending on the payload, the ADC may perform various functions, such as diagnosis, monitoring, and/or therapy. ADCs can be prepared by a variety of methods. However, most of the methods result in heterogeneous mixtures of chemically distinct ADCs with different payload (drug) antibody ratios (DAR) and coupling sites. This heterogeneity can complicate manufacturing, leading to high variability from batch to batch, and sometimes unpredictable safety and efficacy. Thus, methods that can produce homogeneous mixtures (e.g., regioselective or site-specific coupling methods) are of increasing interest. Such methods can significantly improve the predictability of DAR and payload (drug) binding sites and can be used to simplify the development and manufacture of more defined ADC products with more predictable safety or efficacy. Several methods have been developed for the region-specific and site-specific coupling of payloads to antibodies. However, generally known methods require modification/engineering of antibodies, for example by incorporation of unnatural amino acids or by modification of carbohydrate moieties. Such modifications may negatively impact the therapeutic efficacy/safety of the corresponding ADC, for example, because of undesired effects associated with the activity, targeting, metabolism and/or excretion of the antibody, and immune response to the antibody. Other methods involve multiple steps, such as the methods listed in WO 2018/199337. Such multi-step methods can be costly and/or laborious, making them less attractive, and even unsuitable for applications where a rapid and simple antibody modification process is desired (e.g., for point-of-care diagnostic applications). Thus, there remains a need to find alternative methods for the region or site-specific coupling of payloads to antibodies or antibody fragments, particularly methods that do not require engineering of antibodies or antibody fragments prior to this. Furthermore, there is a need to find a method for preparing antibody drug conjugates in as few steps as possible, preferably in one single step. In view of the above, it is an object of the present invention to provide compounds (reactive conjugates) that enable selective coupling of a payload region to an antibody or antibody fragment in one single step without engineering and/or modification of the antibody or antibody fragment prior thereto. It is another object to provide a kit comprising such a compound. It is a further object of the invention to provide a method for preparing a modified antibody or modified antibody fragment (e.g., ADC) that can be used in a method of diagnosing, monitoring, imaging or treating a disease. Disclosure of Invention The present invention provides a compound that is capable of regioselectively attaching a payload to an antibody (e.g., a therapeutic antibody) or an antibody fragment optionally incorporated into an Fc-fusion protein. This zone selective attachment may be accomplished in a single step. The resulting modified antibodies or modified antibody fragments (e.g., ADC or antibody-radionuclide conjugates) can be used in methods of diagnosing, monitoring, imaging, or treating diseases (particularly cancer). The compound (reactive conjugate) of the present invention can be represented by the following formula (1): P-Y-S-V (1) Wherein, the P is the payload; y is a reactive moiety capable of reacting with a side chain of an amino acid (e.g., lysine or cysteine), preferably a moiety capable of reacting with a side chain of lysine; V is a carrier capable of interacting with the crystallizable fragment (fragment crystallizable, f