WO2024105205A1 - Conjugués anticorps-2 médicaments (a2dc) à groupes clivables par voie enzymatique - Google Patents

Conjugués anticorps-2 médicaments (a2dc) à groupes clivables par voie enzymatique Download PDF

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WO2024105205A1
WO2024105205A1 PCT/EP2023/082136 EP2023082136W WO2024105205A1 WO 2024105205 A1 WO2024105205 A1 WO 2024105205A1 EP 2023082136 W EP2023082136 W EP 2023082136W WO 2024105205 A1 WO2024105205 A1 WO 2024105205A1
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amino acid
acid sequence
set forth
sequence set
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Hans-Georg Lerchen
Beatrix Stelte-Ludwig
Anne-Sophie Rebstock
Sarah JOHANNES
Sandra Berndt
Anette Sommer
Christoph Mahlert
Simone Greven
Urs HAGEMANN
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Bayer Aktiengesellschaft
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • This application relates to antibody-drug conjugates, pharmaceutical compositions, processes for preparation thereof, and the use thereof for treating, preventing, or managing diseases and conditions including hyperproliferative disorders, such as, cancer in humans and other mammals.
  • an antibody-drug conjugate of Formula (I), or a pharmaceutically acceptable salt thereof wherein: AK1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur atom of a cysteine, an antibody attached via a nitrogen atom of a lysine, or an antibody attached via both a sulfur atom of a cysteine and via a nitrogen atom of a lysine); X 1 and X 2 are each independently -NH- or -S-; L 1 and L 2 are each independently a stable linker; R A is a first protease-cleavable kinesin spindle protein inhibitor group; R B is a second protease-cleavable kinesin spindle protein inhibitor group; wherein R A and R B are non-identical groups; and a and b are each independently an integer from 1 to 50.
  • AK1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur atom of a
  • an antibody-drug conjugate of Formula (III), or a pharmaceutically acceptable salt thereof ( ) wherein: AK 3 is an antibody or an antigen-binding fragment thereof, attached via a sulfur atom or attached via a nitrogen atom; L 4 is a stable linker; KSP 5 is a first kinesin spindle protein inhibitor; P 5 is protease cleavable linker; KSP6 is a second kinesin spindle protein inhibitor; and y is an integer from 1 to 50.
  • provided herein is a pharmaceutical composition comprising an antibody-drug conjugate described herein, or a pharmaceutically acceptable salt thereof, and an inert non-toxic pharmaceutically suitable auxiliary.
  • a method for the treatment or prophylaxis of diseases the method comprising administering to a patient in need thereof an antibody-drug conjugate described herein.
  • a method for treatment or prophylaxis of hyperproliferative or angiogenic disorders the method comprising administering to a patient in need thereof an antibody-drug conjugate described herein.
  • FIG.1 shows A2DC potency (Ex I-11m) at Mesothelin-transfected HT29 cells and HT29 wild type cells compared to isotype control A2DC and the respective ADC components in the A2DC.
  • FIG.2 shows A2DC retains by-stander killing of permeable ADC component: Potency of A2DC (Ex I-6m) at Mesothelin-transfected HT29 cells, HT29 wild type and co- culture in comparison to the respective ADC components in the A2DC.
  • FIG.3 shows tumor growth of NCI-H292 xenografts after treatment with A2DC.
  • FIG.4 shows tumor growth of CT26 (syngeneic model) after treatment with A2DC
  • FIG.5 shows analysis of different metabolites intracellularly and extracellularly after incubation of mesothelin-transfected HT29 cells with A2DC of Example II-6m.
  • FIG.6 shows tumor growth of CT26 (syngeneic model) after treatment with A2DC. INCORPORATION BY REFERENCE [0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
  • ADC Antibody-drug conjugates
  • ADC are an emerging drug class that utilizes antibodies to improve targeting of cytotoxic or immuno oncology payloads to tumor cells.
  • An active metabolite is formed in most cases upon internalization of the ADC into cancer cells where an active metabolite is formed either by enzymatic cleavage of the linker or by antibody degradation.
  • the approval of meanwhile 12 ADCs demonstrates the potential of ADCs to improve cancer treatment.
  • the drug-to-antibody ratio characterizes the number of payloads which is linked to the antibody.
  • DAR drug-to-antibody ratio
  • a higher DAR in general may lead to a more efficient payload delivery and thus may result in a higher potency of ADCs.
  • an increase of the DAR often results in poor physicochemical properties of the ADC. Consequences are an increased susceptibility of ADCs to aggregate formation, a higher degree of unspecific uptake in non-tumor cells associated with strong side effects and a higher clearance and poor PK parameter of ADCs.
  • ADCs with high drug loading without compromising physicochemical and pharmacokinetic properties (e.g. A. V. Yurkovetskiy in Cancer Research (2015), 75(16), 3365-3372), which however adds another level of complexity to ADCs.
  • Another key aspect for ADC performance depending on the tumor to be treated is the physicochemical profile of the payload which is released.
  • the release of cell-permeable payloads may be associated with a bystander killing effect, which is considered to be particularly beneficial for the treatment of tumors with heterogenous target expression (J. A. Costoplus, ACS Med Chem Lett, 2019, 10, 1393).
  • such a cell-permeable metabolite profile may increase side effects due to the re- distribution of released payload or premature payload cleavage which both causes damage to healthy cells.
  • the release of a non-permeable payloads may result in intracellular retention and accumulation of the active drug inside the tumor cell, which may have a beneficial impact on potency.
  • such a profile may increase safety of the ADCs, as the released non-permeable payload does not induce damage to healthy cells.
  • any bystander killing of the released payload cannot be achieved which such a metabolite design.
  • KSP Kinesin spindle protein
  • Eg5 Kinesin spindle protein
  • KIF11 Kinesin spindle protein
  • the blockade of this essential event in mitosis with small molecule KSP inhibitors (KSPis) results in high antitumor potency which raised great interest in this compound class (H. B. El-Nassan, Eur. J. Med. Chem.2013, 62, 614).
  • SMOL KSPi small molecule KSP inhibitors
  • KSPi payload molecules offer various options for payload attachment which are compatible with either cleavable or with non-cleavable linker chemistries.
  • Legumain is an asparaginyl endopeptidase overexpressed in solid tumors, and it has been associated with invasion, metastasis, and poor survival (Dall, E., and Brandstetter, H. (2016) Biochimie 2016, 22, 126 ⁇ 50).
  • WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578 different ADCs are claimed which are activated by the tumor associated protease legumain to release an active KSPi payload.
  • the profile of the cleaved KSPi payload can be modified and adapted to different physicochemical properties and to achieve different levels of membrane permeability without disrupting the inhibition of the KSP target.
  • Detailed Description of the Invention [0027]
  • each KSP e.g., KSP 1 , KSP 2 , KSP 3 , KSP 4 , KSP 5 , and KSP 6
  • each R e.g., R A , R B , R C , and R D
  • each P e.g., P 1 , P 2 , P 3 , P 4 , and P 5
  • each L e.g., L 1 , L 2 , L 3 , and L 4
  • each AK e.g., AK 1 , AK 2 , and AK 3
  • each AK is an antibody or antigen-binding fragment thereof, wherein the antibody can be monoclonal or
  • the compound is an antibody-drug conjugate of Formula (I), or a pharmaceutically acceptable salt thereof: wherein: AK 1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur atom of a cysteine, an antibody attached via a nitrogen atom of a lysine, or an antibody attached via both a sulfur atom of a cysteine and via a nitrogen atom of a lysine); X 1 and X2 are each independently -NH- or -S-; L 1 and L 2 are each independently a stable linker; R A is a first protease-cleavable kinesin spindle protein inhibitor group; R B is a second protease-cleavable kinesin spindle protein inhibitor group; wherein R A and R B are non-identical groups; and a and b are each independently an integer from 1 to 50.
  • AK 1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur
  • R A is a first protease-cleavable kinesin spindle protein inhibitor of formula (i): (i) KSPi 1 -P 1 - # ; wherein KSPi 1 is a first kinesin spindle protein inhibitor, P 1 is a first protease-cleavable linker; and # is a bond from R A to L 1 ; and R B is a second protease-cleavable kinesin spindle protein inhibitor of formula (ii): (ii) ## -P 2 -KSPi 2 ; wherein ## is a bond from L 2 to R B ; P 2 is a second protease-cleavable linker; KSPi2 is a second kinesin spindle protein inhibitor, and wherein R A and R B are non-identical groups.
  • the antibody-drug conjugate of Formula (I), or a pharmaceutically acceptable salt thereof has the structure of Formula (I’): wherein: AK1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur atom of a cysteine, an antibody attached via a nitrogen atom of a lysine, or an antibody attached via both a sulfur atom of a cysteine and via a nitrogen atom of a lysine); X 1 and X 2 are each independently -NH- or -S-; L 1 and L 2 are each independently: P 1 is a first protease-cleavable linker; P 2 is a second protease-cleavable linker; R 1 and R 6 are each independently halogen; R 2 and R 7 are each independently C 1-6 alkyl; m and n are each independently an integer from 1 to 5; and a and b are each independently an integer from 1 to 50.
  • AK1 is an antibody or antigen binding fragment thereof (
  • P 1 is a first protease-cleavable linker.
  • P 2 is a second protease-cleavable linker.
  • P 1 and P 2 are each independently a cathepsin or legumain-cleavable linker.
  • P 1 and P 2 are each independently a legumain-cleavable linker.
  • P 1 and P 2 are each independently a legumain-cleavable linker comprising one or more of amino acid sequences (1)- (8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.
  • the antibody-drug conjugate is a compound of Formula (I), wherein: AK1 is an antibody or antigen binding fragment thereof (e.g., an antibody attached via a sulfur atom of a cysteine, an antibody attached via a nitrogen atom of a lysine, or an antibody attached via both a sulfur atom of a cysteine and via a nitrogen atom of a lysine); X 1 and X 2 are each independently -NH- or -S-; L 1 and L 2 are each independently: , , wherein: R 1 and R 6 are each independently halogen; R 2 and R 7 are each independently C 1-6 alkyl; R 3 and R 8 are each independently C 1-6 alkyl or -C 1-6 alkylene-COOH; R 4 , R 5 , R 9 and R 10 are each independently hydrogen or C 1-6 alkyl; a and b are each independently an integer from 1 to 50; m and n are each independently an integer from 1 to 5
  • AK1 is Anti-EGFR, Anti-Her2, Anti-TWEAKR, Anti-CD123, or Anti-CXCR5. In some embodiments, AK1 is Anti-EGFR mAb, Anti-Her2 mAb, Anti- TWEAKR mAb, Anti-CD123 mAb, or Anti-CXCR5 mAb.
  • X 1 and X 2 are each -NH-. In some embodiments, X 1 and X 2 are each -S-. In some embodiments, X 1 is -NH- and X 2 is -S-. In some embodiments, X 1 is -S- and X 2 is -NH-.
  • L 1 and L 2 are each independently: . [0037] In some embodiments, L 1 and L 2 are each independently: , , or . [0038] In some embodiments, L 1 and L 2 are each independently: or . [0039] In some embodiments, each R 1 is independently halogen. In some embodiments, each R 1 is chloro, fluoro, or bromo. In some embodiments, each R 1 is independently chloro or fluoro. In some embodiments, each R 1 is independently fluoro. [0040] In some embodiments, each R 2 is independently C 1-6 alkyl. In some embodiments, each R 2 is independently C 3-6 alkyl.
  • each R 2 is independently n-propyl, isopropyl, n-butyl, tert-butyl, or iso-butyl. In some embodiments, each R 2 is independently - C(CH 3 ) 3 . [0041] In some embodiments, each R 3 is independently C 1-6 alkyl or -C 1-6 alkylene-COOH. In some embodiments, each R 3 is independently C 1-3 alkyl or -C 1-3 alkylene-COOH. In some embodiments, each R 3 is independently methyl, ethyl, propyl, or -C 1-3 alkylene-COOH. In some embodiments, each R 3 is independently methyl.
  • each R 3 is independently -CH2COOH.
  • each R 4 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 4 is independently hydrogen or C 1-3 alkyl. In some embodiments, each R 4 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 4 is independently hydrogen or methyl. In some embodiments, each R 4 is hydrogen. In some embodiments, each R 4 is methyl.
  • each R 5 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 5 is independently hydrogen or C 1-3 alkyl. In some embodiments, each R 5 is independently hydrogen, methyl, or ethyl.
  • each R 5 is independently hydrogen or methyl. In some embodiments, each R 5 is hydrogen. In some embodiments, each R 5 is methyl. [0044] In some embodiments, each R 6 is independently halogen. In some embodiments, each R 6 is chloro, fluoro, or bromo. In some embodiments, each R6 is independently chloro or fluoro. In some embodiments, each R 6 is independently fluoro. [0045] In some embodiments, each R 7 is independently C 1-6 alkyl. In some embodiments, each R 7 is independently C 3-6 alkyl. In some embodiments, each R 7 is independently n-propyl, isopropyl, n-butyl, tert-butyl, or iso-butyl.
  • each R 7 is independently - C(CH 3 ) 3 .
  • each R 8 is independently C 1-6 alkyl or -C 1-6 alkylene-COOH. In some embodiments, each R 8 is independently C 1-3 alkyl or -C 1-3 alkylene-COOH. In some embodiments, each R 8 is independently methyl, ethyl, propyl, or -C 1-3 alkylene-COOH. In some embodiments, each R 8 is independently methyl. In some embodiments, each R 8 is independently -CH2COOH. [0047] In some embodiments, each R 9 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 9 is independently hydrogen or C 1-3 alkyl.
  • each R 9 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 9 is independently hydrogen or methyl. In some embodiments, each R 9 is hydrogen. In some embodiments, each R 9 is methyl.
  • each R 10 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 10 is independently hydrogen or C 1-3 alkyl. In some embodiments, each R 10 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 10 is independently hydrogen or methyl. In some embodiments, each R 10 is hydrogen. In some embodiments, each R 10 is methyl. [0049] In some embodiments, a and b are each independently an integer from 1 to 50.
  • a is an integer from 1 to 25. In some embodiments, a is an integer from 1 to 20. In some embodiments, a is an integer from 1 to 15. In some embodiments, a is an integer from 1 to 10. In some embodiments, a is an integer from 1 to 5. In some embodiments, b is an integer from 1 to 25. In some embodiments, b is an integer from 1 to 20. In some embodiments, b is an integer from 1 to 15. In some embodiments, b is an integer from 1 to 10. In some embodiments, b is an integer from 1 to 5. [0050] In some embodiments, m is an integer from 1 to 5. In some embodiments, m is an integer from 1 to 4. In some embodiments, m is an integer from 1 to 3.
  • n is 1 or 2. In some embodiments m is 2. In some embodiments, m is 1. [0051] In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments n is 2. In some embodiments, n is 1. [0052] In some embodiments, p is 0, 1, 2, 3, or 4. In some embodiments, p is 0, 1, 2, or 3. In some embodiments, p is 0, 1, or 2. In some embodiments, p is 0 or 1. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2.
  • p is 3. [0053] In some embodiments, q is 0, 1, 2, 3, or 4. In some embodiments, q is 0, 1, 2, or 3. In some embodiments, q is 0, 1, or 2. In some embodiments, q is 0 or 1. In some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. [0054] In some embodiments, r is 0, 1, 2, 3, or 4. In some embodiments, r is 0, 1, 2, or 3. In some embodiments, r is 0, 1, or 2. In some embodiments, r is 0 or 1. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3.
  • s is 0, 1, 2, 3, or 4. In some embodiments, s is 0, 1, 2, or 3. In some embodiments, s is 0, 1, or 2. In some embodiments, s is 0 or 1. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. [0056] In some embodiments, the R A -L 1 -X 1 - fragment is:
  • -X 2 -L 2 -R B fragment is:
  • the antibody-drug conjugate of Formula (I) has a structure of Formula (IA), or a pharmaceutically acceptable salt thereof: [0059] In some embodiments, the antibody-drug conjugate is:
  • the antibody-drug conjugate of Formula (I) has a structure of Formula (IB), or a pharmaceutically acceptable salt thereof:
  • the antibody-drug conjugate is: .
  • the antibody-drug conjugate of Formula (I) has a structure of Formula (IC), or a pharmaceutically acceptable salt thereof:
  • the antibody-drug conjugate is:
  • the compound is an antibody-drug conjugate of Formula (II), or a pharmaceutically acceptable salt thereof: wherein: AK 2 is an antibody attached via a sulfur atom or attached via a nitrogen atom; L 3 is a stable linker; R C and R D are each independently a protease-cleavable kinesin spindle protein inhibitor group; and z is an integer from 1 to 50.
  • the antibody-drug conjugate of Formula (II), or a pharmaceutically acceptable salt thereof has the structure of Formula (II’): wherein: AK 2 is an antibody attached via a sulfur atom of a cysteine, attached via a nitrogen atom of a lysine, or a combination thereof; L3 is a stable linker; P 3 and P 4 are each independently a protease-cleavable linker; KSPi 3 and KSPi 4 are each independently a kinesin spindle protein inhibitor; and z is an integer between 1 and 50.
  • KSPi 3 and KSPi 4 are each independently: wherein: each R 11 is halogen; and n is an integer from 1 to 5.
  • P 3 and P 4 are each independently a protease-cleavable linker.
  • P 3 and P 4 are each independently a cathepsin or legumain-cleavable linker.
  • P 3 and P 4 are each independently a legumain-cleavable linker.
  • P 3 and P 4 are each independently a legumain-cleavable linker comprising one or more of amino acid sequences (1)-(8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.
  • AK 2 is Anti-EGFR, Anti-Her2, Anti-TWEAKR, Anti-CD123, or Anti-CXCR 5 .
  • AK 2 is Anti-EGFR mAb, Anti-Her2 mAb, Anti- TWEAKR mAb, Anti-CD123 mAb, or Anti-CXCR 5 mAb.
  • L3 is: , , , ,
  • each R 11 is independently halogen. In some embodiments, each R 11 is chloro, fluoro, or bromo. In some embodiments, each R 11 is independently chloro or fluoro. In some embodiments, each R 11 is independently fluoro.
  • each R 12 is independently C 1-6 alkyl or -C 1-6 alkylene-COOH. In some embodiments, each R 12 is independently C 1-3 alkyl or -C 1-3 alkylene-COOH. In some embodiments, each R 12 is independently methyl, ethyl, propyl, or -C 1-3 alkylene-COOH. In some embodiments, each R 12 is independently methyl.
  • each R 12 is independently -CH 2 COOH.
  • each R 13 is independently C 1-6 alkyl. In some embodiments, each R 13 is independently C 1-3 alkyl. In some embodiments, each R 13 is independently methyl, ethyl, n-propyl, or iso-propyl. In some embodiments, each R 13 is independently methyl, n- propyl, or iso-propyl. In some embodiments, each R 13 is methyl. In some embodiments, each R 13 is iso-propyl. [0074] In some embodiments, each R 14 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 14 is independently hydrogen or C 1-3 alkyl.
  • each R 14 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 14 is independently hydrogen or methyl. In some embodiments, each R 14 is hydrogen. In some embodiments, each R 14 is methyl. [0075] In some embodiments, each R 15 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 15 is independently hydrogen or C 1-3 alkyl. In some embodiments, each R 15 is independently hydrogen, methyl, ethyl, or propyl. In some embodiments, each R 15 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 15 is independently hydrogen or methyl. In some embodiments, each R 15 is hydrogen. In some embodiments, each R 15 is methyl.
  • each R 16 is independently C 1-6 alkyl or -C 1-6 alkylene-COOH. In some embodiments, each R 16 is independently C 1-6 alkyl. In some embodiments, each R 16 is - C 1-6 alkylene-COOH. In some embodiments, each R 16 is -C 1-5 alkylene-COOH. In some embodiments, each R 16 is -C 1-4 alkylene-COOH. In some embodiments, each R 16 is independently -C 1-3 alkylene-COOH. In some embodiments, each R 16 is -C 1-2 alkylene-COOH. In some embodiments, each R 16 is -CH 2 COOH.
  • each R 17 is independently hydrogen or C 1-6 alkyl. In some embodiments, each R 17 is independently hydrogen or C 1-3 alkyl. In some embodiments, each R 17 is independently hydrogen, methyl, ethyl, or propyl. In some embodiments, each R 17 is independently hydrogen, methyl, or ethyl. In some embodiments, each R 17 is independently hydrogen or methyl. In some embodiments, each R 17 is hydrogen. In some embodiments, each R 17 is methyl. [0078] In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2.
  • n is 2. In some embodiments, n is 1. [0079] In some embodiments, r is 0, 1, 2, 3, or 4. In some embodiments, r is 0, 1, 2, or 3. In some embodiments, r is 0, 1, or 2. In some embodiments, r is 0 or 1. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. [0080] In some embodiments, s is 0, 1, 2, 3, or 4. In some embodiments, s is 0, 1, 2, or 3. In some embodiments, s is 0, 1, or 2. In some embodiments, s is 0 or 1. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, s is 2.
  • s is 3. [0081] In some embodiments, t is 0, 1, 2, 3, or 4. In some embodiments, t is 0, 1, 2, or 3. In some embodiments, t is 0, 1, or 2. In some embodiments, t is 0 or 1. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, s is 2. In some embodiments, t is 3. [0082] In some embodiments, RC is:
  • R D is:
  • the antibody-drug conjugate is:
  • the compound is an antibody-drug conjugate of Formula (III), or a pharmaceutically acceptable salt thereof: (III) wherein: AK 3 is an antibody or an antigen-binding fragment thereof, attached via a sulfur atom or attached via a nitrogen atom; L 4 is a stable linker; KSP 5 is a first kinesin spindle protein inhibitor; P 5 is protease cleavable linker; KSP 6 is a second kinesin spindle protein inhibitor; and y is an integer from 1 to 50.
  • Formula (III) wherein: AK 3 is an antibody or an antigen-binding fragment thereof, attached via a sulfur atom or attached via a nitrogen atom; L 4 is a stable linker; KSP 5 is a first kinesin spindle protein inhibitor; P 5 is protease cleavable linker; KSP 6 is a second kinesin spindle protein inhibitor; and y is an integer from 1 to 50.
  • L 4 is a substituted or unsubstituted C 1-60 alkyl linker, or a substituted or unsubstituted 1-60 membered heteroalkyl linker.
  • L 4 has the structure: wherein #AK 3 denotes a bond from L 4 to AK 3 ; and # denotes a bond from L 4 to KSPi 5 .
  • the antibody-drug conjugate is a compound of Formula (III) wherein: KSPi 5 is a first kinesin spindle protein inhibitor having the structure: KSPi 6 is a second kinesin spindle protein inhibitor having the structure: wherein: # denotes a bond from L 4 to KSPi 5 ; ## denotes a bond from KSPi 5 to P 5 ; ### denotes a bond from P 5 to KSPi 6 ; each R 21 is independently halogen; each R 22 is independently C 1-6 alkyl; and n is an integer from 1 to 5. [0088] In some embodiments, P 5 is a protease-cleavable linker.
  • P 5 is a cathepsin or legumain-cleavable linker. In some embodiments, P 5 is a legumain-cleavable linker. In some embodiments, P 5 is a legumain-cleavable linker comprising one or more of amino acid sequences (1)-(8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8)
  • P 5 is a legumain-cleavable linker having the structure: -P 5a -L 5 -P 5b - wherein: P 5a and P 5b are each independently protease cleavable linkers; and L 5 is a stable linker.
  • P 5a and P 5b are each independently a legumain-cleavable linker comprising one or more of amino acid sequences (1)-(8): (1) -(L-Asn)(NMe-L-Ala)(L-Ala)-, (2) -(L-Asn)(L-Ala)(L-Ala)-, (3) -(L-Asn)(D-Ala)(L-Ala)-, (4) -(L-Asn)(L-Asp)(L-Ala)-, (5) -(L-Asn)(D-Asp)(L-Ala)-, (6) -(L-Asn)(D-Ser)(L-Ala)-, (7) -(L-Asn)(L-Ala)-, or (8) -(L-Asn)-.
  • L5 is a stable linker that is a substituted or unsubstituted C 1-60 alkyl linker, or a substituted or unsubstituted 1-60 membered heteroalkyl linker.
  • L 5 is a stable linker that is a substituted or unsubstituted C 1-50 alkyl linker, or a substituted or unsubstituted 1-50 membered heteroalkyl linker.
  • L5 is a stable linker that is a substituted or unsubstituted C 1-40 alkyl linker, or a substituted or unsubstituted 1-40 membered heteroalkyl linker.
  • L5 is a stable linker that is a substituted or unsubstituted C 1-30 alkyl linker, or a substituted or unsubstituted 1-30 membered heteroalkyl linker. In some embodiments, L5 is a stable linker that is a substituted or unsubstituted C 1-20 alkyl linker, or a substituted or unsubstituted 1-20 membered heteroalkyl linker. In some embodiments, L 5 is a stable linker that is a substituted or unsubstituted C 1-10 alkyl linker, or a substituted or unsubstituted 1-10 membered heteroalkyl linker.
  • P 5 is a legumain-cleavable linker having the structure: [0093]
  • AK 3 is Anti-EGFR, Anti-Her2, Anti-TWEAKR, Anti-CD123, or Anti-CXCR 5 .
  • AK 3 is Anti-EGFR mAb, Anti-Her2 mAb, Anti- TWEAKR mAb, Anti-CD123 mAb, or Anti-CXCR 5 mAb.
  • each R 21 is independently halogen. In some embodiments, each R 21 is chloro, fluoro, or bromo. In some embodiments, each R 21 is independently chloro or fluoro.
  • each R 21 is independently fluoro.
  • each R 22 is independently C 1-6 alkyl. In some embodiments, each R 22 is independently C 1-4 alkyl. In some embodiments, each R 22 is independently n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl. In some embodiments, each R 22 is independently tert-butyl. [0096] In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is 1 or 2. In some embodiments n is 2.
  • n is 1.
  • y is an integer from 1 to 50. In some embodiments, y is an integer from 1 to 25. In some embodiments, y is an integer from 1 to 20. In some embodiments, y is an integer from 1 to 15. In some embodiments, y is an integer from 1 to 10. In some embodiments, y is an integer from 1 to 6. In some embodiments, y is an integer from 2 to 6. [0098] In some embodiments, KSPi 5 is: ; and
  • KSPi 5 is:
  • the anti-body drug conjugate is: ,
  • the Antibody-2-drug conjugates of the current invention are obtained by subsequent coupling of 2 different payload linker precursors to the same antibody following the descriptions described in WO2016/207089, WO2017/162663, WO2018/114798 and applied independently twice.
  • the resulting A2DCs of the current invention comprise two different payload linkers, both attached to lysine residues, or two different payload linkers, both attached to cysteine residues or one payload linker attached to lysine residues and the other payload linker attached to cysteine residues.
  • the cysteine residues suitable for payload linker attachment can be obtained by reduction of interchain disulfide bridges or by engineering of cysteine residues into the antibody.
  • the A2DCs of the current invention with different antibodies targeting a variety of targets combine different payload features resulting in a favorable profile: – Overcome the big challenge of increasing the DAR without running into aggregate formation, in particular due to polar and hydrophilic KSPi payloads. – Non-permeable payloads usually increase potency due to intracellular accumulation in tumor cells, particularly beneficial in vivo when tumor cells show a homogenous target expression. A very high potency is achieved when two non-permeable KSPi payloads are linked to the antibody resulting in an increased DAR.
  • A2DCs of the current invention combine positive features which provides efficacious A2DCs against different cells with a broad variability of target expression profiles and are shown to be compatible with antibodies addressing different targets [0103]
  • antibody conjugates have been provided with peptide derivatives which can be released by tumour-associated enzymes such as legumain or cathepsin. The tumour selectivity is thus determined not just by the choice of antibody but additionally by the enzymatic cleavage of the peptide derivative, for example by the tumour-associated enzyme legumain.
  • the peptide derivative may be present in the linker which connects the antibody to the KSP inhibitor.
  • These are the antibody-2-drug conjugates (A2DCs) according to the invention.
  • A2DCs antibody-2-drug conjugates
  • the kinesin spindle protein inhibitors used in accordance with the invention have an amino group which is essential to the effect. By modification of this amino group with peptide derivatives, the effect with respect to the kinesin spindle protein is blocked and hence the development of a cytotoxic effect is also inhibited. If this peptide residue, however, can be released by tumour-associated enzymes such as legumain, the effect can be re-established in a controlled manner in the tumour tissue. The modification of the amino group in this case is not part of the linker.
  • the present invention relates to binder conjugates having inactive precursor molecules of the kinesin spindle protein inhibitors which are only processed in the tumour by means of the tumour-associated lysosomal endopeptidase legumain to give the active metabolites, in order thus to be able to display their cytotoxic activity again in a controlled manner in the tumour.
  • the binder conjugates with KSP inhibitors, wherein the free amino group thereof is correspondingly blocked, are also referred to in accordance with the invention as APDCs.
  • the APDCs are particularly preferred.
  • the Antibody-2-drug conjugates of the current invention have enzymatically cleavable peptide sequences P 3 and P 4 capable to release two active metabolites which can be tailored with respect to a desired physicochemical profile in a flexible manner.
  • the attachment to the antibody is accomplished via a branched linker L3
  • the attachment to the antibody is accomplished via a non- cleavable linker L 4 , which is attached to the KSPi 5 .
  • the A2DCs can be attached to the antibody im multiple ways, either via chemical or enzymatic attachment protocols, preferably via lysine residues or via cysteine residues.
  • the cysteine residues suitable for payload linker attachment can be obtained by reduction of interchain disulfide bridges or by engineering of cysteine residues into the antibody.
  • the A2DCs of the current invention with different antibodies targeting a variety of targets combine different payload features resulting in a favorable profile: – Overcome the big challenge of increasing the DAR without running into aggregate formation, in particular by utilizing polar and hydrophilic KSPi payloads and linker compositions.
  • Non-permeable payloads usually increase potency due to intracellular accumulation in tumor cells, which is particularly beneficial in vivo when tumor cells show a homogenous target expression.
  • a very high potency is achieved when two non-permeable KSPi payloads are linked to the antibody resulting in an increased DAR.
  • Permeable payload display a by-stander effect, which is particularly important when tumors show heterogenous target expression
  • A2DCs of the current invention combine positive features which provides efficacious A2DCs against different cells with a broad variability of target expression profiles. This is shown to be compatible with antibodies addressing different targets.
  • the release of both metabolites is mediated by linker cleavage by the tumor associated proteases legumain.
  • the A2DCs can also be designed for payload release mediated by other proteases such as cathepsins or by glycosidases such as ß-glucuronidase.
  • the target molecule is a selective cancer target molecule.
  • the target molecule is a protein.
  • the target molecule is an extracellular target molecule.
  • the extracellular target molecule is a protein. Cancer target molecules are known to those skilled in the art. Examples of these are listed below.
  • cancer target molecules are: (1) EGF receptor (NCBI reference sequence NP_005219.2), SEQ ID NO: 213 (1210 amino acids. (2) Mesothelin (SwissProt reference Q13421-3), SEQ ID NO: 214 (622 amino acids).
  • the antibodies of the present invention can be any suitable antibody.
  • the antibodies include, but are not limited to GUCY2C (guanylate cyclase C), ROR1, HER 3 , FGFR 4 , EGFR/PD-L1 bispecific, and B7H4.
  • Polyclonal antibodies can be prepared by methods known to a person of ordinary skill in the art.
  • Monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Köhler and Milstein, Nature, 256, 495-497, 1975).
  • Human and humanized monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Olsson et al., Meth Enzymol.92, 3-16 or Cabilly et al U.S. Pat. No.4,816,567 or Boss et al U.S. Pat. No.4,816,397).
  • AK antibody or antigen binding fragment thereof
  • AK comprises an antibody or antibody region (e.g., CDR, variable domain) shown in the table below:
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 3; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 4; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 6; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 7; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 8.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 heavy chain complementarity determining region 2
  • HCDR3 comprising
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 12; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 13; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 14; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 32; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 33; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 34; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 62; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 63; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 64; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in SEQ
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 72; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 73; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 74; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in SEQ
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 82; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 83; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 84; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in S
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 92; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 93; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 94; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 96; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 97; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 98.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in S
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 106; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 107; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 108.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in S
  • the antibody or antigen binding fragment thereof comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 112; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 113; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 114; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 116; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 117; and/or f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 118.
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 comprising the amino acid sequence set forth in S
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 5.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 9; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 10.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 10.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 11; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 15.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 19; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 20.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 31; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 35.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 35.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 39; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 40.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 40.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 61; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 61; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 65.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 69; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 70.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 69; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 70.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 71; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 75.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 71; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 75.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 79; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 80.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 79; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 80.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 81; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 85.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 81; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 89; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 90.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 89; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 90.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 91; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 95.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 99; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 100.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 99; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 100.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 101; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 105.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 109; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 110.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 109; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 110.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 111; and/or b) an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 115.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; and/or b) an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 119; and/or b) an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 120.
  • the antibody or antigen binding fragment thereof comprises: a) an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 119; and/or b) an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 120.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Definitions [0144] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains.
  • the terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context. [0148]
  • the terms “subject,” “individual,” or “patient” are often used interchangeably herein.
  • a “subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • the term “about” a number refers to that number plus or minus 15% of that number.
  • the term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.
  • the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • cancers are the consequence of uncontrolled cell growth of the most diverse tissues. In many cases the new cells penetrate into existing tissue (invasive growth), or they metastasize into remote organs. Cancers occur in a wide variety of different organs and often have tissue- specific courses.
  • the term “cancer” as a generic term therefore describes a large group of defined diseases of different organs, tissue and cell type.
  • the term “cancer target molecule” describes a target molecule which is more abundantly present on one or more cancer cell species than on non-cancer cells of the same tissue type.
  • the cancer target molecule is selectively present on one or more cancer cell species compared with non-cancer cells of the same tissue type, where selectively describes an at least two-fold enrichment on cancer cells compared to non-cancer cells of the same tissue type (a “selective cancer target molecule”).
  • a “selective cancer target molecule” allows the selective therapy of cancer cells using the conjugates according to the invention.
  • a “target molecule” in the broadest sense is understood to mean a molecule which is present in the target cell population and which may be a protein (for example a receptor of a growth factor) or a non-peptidic molecule (for example a sugar or phospholipid). It is preferably a receptor or an antigen.
  • extracellular target molecule describes a target molecule, attached to the cell, which is located at the outside of a cell, or the part of a target molecule which is located at the outside of a cell, i.e. a binder may bind on an intact cell to its extracellular target molecule.
  • An extracellular target molecule may be anchored in the cell membrane or be a component of the cell membrane.
  • the person skilled in the art is aware of methods for identifying extracellular target molecules. For proteins, this may be by determining the transmembrane domain(s) and the orientation of the protein in the membrane. These data are usually deposited in protein databases (e.g. SwissProt).
  • an immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains) which are typically linked by disulfide bridges.
  • Each heavy chain comprises a variable domain of the heavy chain (abbreviated VH) and a constant domain of the heavy chain.
  • the constant domain of the heavy chain may, for example, comprise three domains CH1, CH2 and CH3.
  • Each light chain comprises a variable domain (abbreviated VL) and a constant domain.
  • the constant domain of the light chain comprises a domain (abbreviated CL).
  • CL constant domain
  • the VH and VL domains may be subdivided further into regions having hypervariability, also referred to as complementarity determining regions (abbreviated CDR) and regions having low sequence variability (framework region, abbreviated FR).
  • CDR complementarity determining regions
  • FR frame region
  • each VH and VL region is composed of three CDRs and up to four FRs.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 For example from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • An antibody may be obtained from any suitable species, e.g. rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin.
  • An antibody may, for example, be human, humanized or chimeric.
  • the term “monoclonal” antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e. individual antibodies of the population are identical except for naturally occurring mutations, of which there may be a small number. Monoclonal antibodies recognize a single antigenic binding site with high specificity. The term monoclonal antibody does not refer to a particular preparation process. [0157] Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies may be categorized into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be divided into further subclasses.
  • the constant domains of the heavy chain which correspond to the different classes, are referred to as [alpha/ ⁇ ], [delta/ ⁇ ], [epsilon/ ⁇ ], [gamma/ ⁇ ] and [my/ ⁇ ]. Both the three-dimensional structure and the subunit structure of antibodies are known.
  • the term “intact” antibody refers to antibodies comprising both an antigen-binding domain and the constant domain of the light and heavy chain.
  • the constant domain may be a naturally occurring domain or a variant thereof having a number of modified amino acid positions, and may also be a glycosylated.
  • modified intact antibody refers to intact antibodies fused via their amino terminus or carboxy terminus by means of a covalent bond (e.g. a peptide bond) with a further polypeptide or protein not originating from an antibody.
  • antibodies may be modified such that, at defined positions, reactive cysteines are introduced to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol.2008 August; 26(8):925-32).
  • “Amino acid modification” or “mutation” here means an amino acid substitution, insertion and/or deletion in a polypeptide sequence. The preferred amino acid modification here is a substitution.
  • amino acid substitution or “substitution” here means an exchange of an amino acid at a given position in a protein sequence for another amino acid.
  • substitution Y50W describes a variant of a parent polypeptide in which the tyrosine at position 50 has been exchanged for a tryptophan.
  • a “variant” of a polypeptide describes a polypeptide having an amino acid sequence substantially identical to a reference polypeptide, typically a native or “parent” polypeptide.
  • the polypeptide variant may have one or more amino acid exchanges, deletions and/or insertions at particular positions in the native amino acid sequence.
  • the term “human” antibody refers to antibodies which can be obtained from a human or which are synthetic human antibodies.
  • a “synthetic” human antibody is an antibody which is partially or entirely obtainable in silico from synthetic sequences based on the analysis of human antibody sequences.
  • a human antibody can be encoded, for example, by a nucleic acid isolated from a library of antibody sequences of human origin. An example of such an antibody can be found in Soderlind et al., Nature Biotech.2000, 18:853-856.
  • Such “human” and “synthetic” antibodies also include aglycosylated variants which have been produced either by deglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) of the heavy chain to any other amino acid.
  • humanized or “chimeric” antibody describes antibodies consisting of a non-human and a human portion of the sequence. In these antibodies, part of the sequences of the human immunoglobulin (recipient) is replaced by sequence portions of a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, amino acids of the CDR of the recipient are replaced by amino acids of the donor. Sometimes, amino acids of the framework, too, are replaced by corresponding amino acids of the donor. In some cases the humanized antibody contains amino acids present neither in the recepient nor in the donor, which were introduced during the optimization of the antibody.
  • variable domains of the donor immunoglobulin are fused with the constant regions of a human antibody.
  • Such “humanized” and “chimeric” antibodies also include aglycosylated variants which have been produced either by deglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) of the heavy chain to any other amino acid.
  • the term “complementarity determining region (CDR)” as used herein refers to those amino acids of a variable antibody domain which are required for binding to the antigen. Typically, each variable region has three CDR regions referred to as CDR1, CDR2 and CDR3.
  • Each CDR region may embrace amino acids according to the definition of Kabat and/or amino acids of a hypervariable loop defined according to Chotia.
  • the definition according to Kabat comprises, for example, the region from about amino acid position 24-34 (CDR1), 50-56 (CDR2) and 89-97 ( CDR3) of the variable light chain/domain (VL) and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) of the variable heavy chain/domain (VH) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the definition according to Chotia comprises, for example, the region from about amino acid position 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) of the variable light chain (VL) and 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavy chain (VH) (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)).
  • a CDR may comprise amino acids from a CDR region defined according to Kabat and Chotia.
  • the term “functional fragment” or “antigen-binding antibody fragment” of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g. the variable domains of an IgG) which still comprise the antigen binding domains of the antibody/immunoglobulin.
  • the “antigen binding domain” of an antibody typically comprises one or more hypervariable regions of an antibody, for example the CDR, CDR2 and/or CDR3 region.
  • the “framework” or “skeleton” region of an antibody may also play a role during binding of the antibody to the antigen.
  • the framework region forms the skeleton of the CDRs.
  • the antigen binding domain comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, especially preferably the complete variable light and heavy chains, i.e. amino acids 1-109 of the VL and 1 to 113 of the VH (numbering according to WO97/08320).
  • “Functional fragments” or “antigen-binding antibody fragments” of the invention encompass, non-conclusively, Fab, Fab′, F(ab′)2 and Fv fragments, diabodies, Single Domain Antibodies (DAbs), linear antibodies, individual chains of antibodies (single-chain Fv, abbreviated to scFv); and multispecific antibodies, such as bi- and tri-specific antibodies, for example, formed from antibody fragments C.
  • Multispecific antibodies are those having identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol.1476069; U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol.14815471553).
  • An F(ab′)2 or Fab molecule may be constructed such that the number of intermolecular disulfide interactions occurring between the Ch1 and the CL domains can be reduced or else completely prevented.
  • Epitopic determinants refer to protein determinants capable of binding specifically to an immunoglobulin or T cell receptors. Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains or combinations thereof, and usually have specific 3-dimensional structural properties and also specific charge properties.
  • “Functional fragments” or “antigen-binding antibody fragments” may be fused with another polypeptide or protein, not originating from an antibody, via the amino terminus or carboxyl terminus thereof, by means of a covalent bond (e.g.
  • antibodies and antigen-binding fragments may be modified by introducing reactive cysteines at defined locations, in order to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol.2008 August; 26(8):925-32).
  • An “isolated” antibody or binder has been purified to remove other constituents of the cell. Contaminating constituents of a cell which may interfere with a diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptidic or non-peptidic constituents of a cell.
  • a preferred antibody or binder is one which has been purified to an extent of more than 95% by weight, relative to the antibody or binder (determined for example by Lowry method, UV-Vis spectroscopy or by SDS capillary gel electrophoresis). Moreover an antibody which has been purified to such an extent that it is possible to determine at least 15 amino acids of the amino terminus or of an internal amino acid sequence, or which has been purified to homogeneity, the homogeneity being determined by SDS-PAGE under reducing or non-reducing conditions (detection may be determined by means of Coomassie Blue staining or preferably by silver coloration). However, an antibody is normally prepared by one or more purification steps.
  • the term “specific binding” or “binds specifically” refers to an antibody or binder which binds to a predetermined antigen/target molecule.
  • Specific binding of an antibody or binder typically describes an antibody or binder having an affinity of at least 10 ⁇ 7 M (as Kd value; i.e. preferably those with Kd values smaller than 10 ⁇ 7 M), with the antibody or binder having an at least two times higher affinity for the predetermined antigen/target molecule than for a non-specific antigen/target molecule (e.g. bovine serum albumin, or casein) which is not the predetermined antigen/target molecule or a closely related antigen/target molecule.
  • Kd value i.e. preferably those with Kd values smaller than 10 ⁇ 7 M
  • Specific binding of an antibody or binder does not exclude the antibody or binder binding to a plurality of antigens/target molecules (e.g. orthologs of different species).
  • the antibodies preferably have an affinity of at least 10 ⁇ 7 M (as Kd value; in other words preferably those with smaller Kd values than 10 ⁇ 7 M), preferably of at least 10 ⁇ 9 M, more preferably in the range from 10 ⁇ 9 M to 10 ⁇ 11 M.
  • Kd values may be determined, for example, by means of surface plasmon resonance spectroscopy.
  • Alkyl is a linear or branched saturated monovalent hydrocarbon radical having 1 to 10 carbon atoms (C 1 -C 10 -alkyl), generally 1 to 6 (C 1 -C 6 -alkyl), preferably 1 to 4 (C 1 -C 4 -alkyl) and more preferably 1 to 3 carbon atoms (C 1 -C 3 -alkyl).
  • Preferred examples include: methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2- dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2- methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, and 1,2-dimethylbutyl.
  • Heteroalkyl is a straight-chain and/or branched hydrocarbon chain which has 1 to 10 carbon atoms and may be interrupted once or more than once by one or more of the groups — O—, —S—, —C( ⁇ O)—, —S( ⁇ O)—, —S( ⁇ O)2—, —NR y —, —NR y C( ⁇ O)—, —C( ⁇ O)— NRy—, —NR y NR y —, —S( ⁇ O)2—NR y NR y —, —C( ⁇ O)—NR y NR y —, —CR x ⁇ N—O—, and where the hydrocarbon chain including the side chains, if present, may be substituted by — NH—C( ⁇ O)—NH 2 , —C( ⁇ O)—OH, —OH, —NH 2 , —NH—C( ⁇ NNH 2 )—, sulphon
  • Ry in each case is —H, phenyl, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl or C 2 - C 10 -alkynyl, which may in turn be substituted in each case by —NH—C( ⁇ O)—NH2, — C( ⁇ O)—OH, —OH, —NH 2 , —NH—C( ⁇ NNH 2 )—, sulphonamide, sulphone, sulphoxide, or sulphonic acid.
  • R x is —H, C1-C3-alkyl or phenyl.
  • Alkenyl is a straight-chain or branched monovalent hydrocarbon chain having one or two double bonds and 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms (C 2 -C 10 -alkenyl), especially 2 or 3 carbon atoms (C 2 -C 3 -alkenyl), where, as will be apparent, when the alkenyl group contains more than one double bond, the double bonds may be isolated from one another or conjugated to one another.
  • the alkenyl group is, for example, an ethenyl (or vinyl), prop-2-en-1-yl (or “allyl”), prop-1-en-1-yl, but-3-enyl, but-2-enyl, but-1-enyl, pent-4-enyl, pent-3-enyl, pent-2-enyl, pent-1- enyl, hex-5-enyl, hex-4-enyl, hex-3-enyl, hex-2-enyl, hex-1-enyl, prop-1-en-2-yl (or “isopropenyl”), 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, 1-methylprop- 1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, 2- methylbut-2-enyl, 2- methylbut-2-eny
  • Alkynyl is a straight-chain or branched monovalent hydrocarbon chain having one triple bond and having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms (C2-C10-alkynyl), especially 2 or 3 carbon atoms (C 2 -C 3 -alkynyl).
  • the C 2 -C 6 -alkynyl group is, for example, an ethynyl, prop-1- ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1- methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut- 1-ynyl, 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl, 1-methylpent-4-
  • alkynyl group is ethynyl, prop-1-ynyl or prop-2-ynyl.
  • the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
  • EXAMPLES [0178] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way Part I: Formula I Compounds & Synthesis Preparation and Characterization of Antibody-2-Drug Conjugates (A2DCs) B-1.
  • DNA-sequences that encode the protein sequences (amino acid sequences) of the used antibodies were inserted into a transient expression vector as described by Tom et ah, Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.
  • TPP-981, TPP-1015, TPP-7007, TPP-2658, TPP-2090, TPP-9476, and TPP-9574 were produced in transient mammalian cell cultures as described by Tom et ah, Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.
  • the antibodies for example TPP-981, TPP-1015, TPP-7007, TPP-2658, TPP-2090, TPP-9476, and TPP-9574, were obtained from the cell culture supernatants.
  • the cell supernatants were clarified by centrifugation of cells.
  • the cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. To this end, the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with about 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride.
  • the antibodies were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.
  • the commercially available antibodies were purified from the commercial products by standard chromatography methods (protein A chromatography, preparative gel filtration chromatography (SEC—size exclusion chromatography)).
  • free cysteine residues can be site-specifically engineered into an antibody.
  • the coupling reactions with the electrophiles described in the section ‘ADC precursor molecules‘ were usually carried out under argon as described in the general procedures in the section ‘A2DC examples‘.
  • concentrations of the final A2DC samples were optionally adjusted to the range of 0.5-15 mg/ml by redilution.
  • the respective protein concentrations, stated in the working examples, of the A2DC solutions were determined.
  • antibody loading drug/mAb ratio
  • the A2DCs shown in the examples may also be present to a lesser or higher degree in the form of the hydrolysed open-chain succinamides attached to the antibodies.
  • the KSP-I-A2DCs attached through the linker substructure to thiol groups of the antibodies may optionally also be hydrolized after the coupling by rebuffering and stirring at pH 8 for about 20-24 h to obtain A2DCs attached via open-chain succinamides.
  • #1 represents the sulphur bridge to the antibody, and #2 the point of attachment to the modified KSP inhibitor.
  • Such A2DCs where the linker is attached to the antibodies through hydrolysed open- chain succinamides show higher stability towards de-conjugation by retro-Michael reaction and can be prepared as shown in the scheme below and described in General Procedure A (Lerchen et al., Angew. Chem. Int.
  • AK 1 can mean: Examples a: Anti-EGFR mAb (Cetuximab) (TPP-981), partially reduced Examples e: Anti-Her2 mAb (TPP-1015), partially reduced Examples k-7007: Anti-TWEAKR mAb (TPP-7007), partially reduced Examples k-2658: Anti-TWEAKR mAb (TPP-2658), partially reduced Examples k-2090: Anti-TWEAKR mAb (TPP-2090), partially reduced Examples c: Anti-CD123 mAb (TPP-9476), partially reduced Examples x: Anti-CXCR 5 mAb (TPP-9574), partially reduced where ⁇ 1
  • A2DCs and A2DCs have been characterized by mass spectrometric analysis.
  • Molecular weight analysis was performed using a combination of HPLC and ESI-Q- TOF consisting of e.g. an I-class HPLC (Waters) for sample desalting and separation and an Impact HD mass spectrometer (Bruker Daltonik, Bremen) equipped with instrument control and acquisition software HyStar 3.2, ESI Compass 1.7 and Maximum Entropy Deconvolution Option for MS analysis.
  • Chromatographic system and conditions column: Acquity UPLC BEH300C4 1.7 ⁇ m, 1.0x50mm; column temperature: 70°C; flow: 200 ⁇ l/min; mobile phase solution A: 0.1% formic acid, 94,9% water, 5% ACN; mobile phase solution B: 0.1% formic acid, 9.9% water, 10% ACN and 80% 2-propanol.
  • Binary gradient profile for complete antibody 2min 5% B, 2,5min 50% B, 3,5min 50% B, 5min 95%B, 5,1min 5% B, 5,6min 95% B, 5,7min 5% B, 6,2min 95% B, 6,3min 5% B, 7,5min 5% B
  • Binary gradient profile for reduced antibody 2min 5% B, 4min 30% B, 5min 50% B, 7,5min 50%B, 8,5min 95% B, 8,6min 5% B, 9,1min 95% B, 9,3min 5% B, 9,8min 95% B, 9,9min 5% B, 12min 5% B
  • Cys-coupled ADCs and A2DCs determination of the molecular weights of the individual conjugate species have been measured after deglycosylation and reduction.
  • the cooled sample was acidified with 2 ⁇ l of 10% formic acid/water and then analyzed by mass spectrometry as described above. For DAR determination, all spectra over the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution of light and heavy chain.
  • Average loading of the antibody with toxophors was calculated from the peak areas determined by integration as double the sum of the HC-Load and the LC-load, whereas the HC-load is the sum of toxophor number weighted integration results of all heavy chain (HC) -peaks divided by the sum of the singly weighted integration results of the HC- peaks and whereas the LC-load is the sum of toxophor number weighted integration results of the light chain (LC)-peaks divided by the sum of the singly weighted integration results of all LC peaks.
  • DAR for each toxophore species was calculated separately. The total DAR was calculated as the sum of the individual DARs of both toxophore species.
  • DAR determination For DAR determination, all spectra over the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution of the complete antibody with different numbers of toxophores. The DAR was calculated by dividing the sum of the toxophor number weighted peak area by the sum of the unweighted peak area. For A2DCs, DARs for each toxophore species were calculated separately. The total DAR was calculated as the sum of the individual DARs of both toxophore species.
  • A2DCs composed of a combination of a Cys and a Lys coupled toxophore
  • molecular weights of the individual conjugate species were measured after deglycosylation and reduction.
  • About 160 pmol of the conjugate was diluted with 20mM sodium phosphate pH6.5 in a final volume of 25 ⁇ l.1 ⁇ l PNGase F was added and the sample was incubated overnight at 37°C under gentle shaking (Thermomixer).10 ⁇ l of the deglycosylated sample were denatured by 20 ⁇ l 5M Gu*HCl (Guanidine hydrochloride in 50mM Tri- ethylammoniumhydrogencarbonat) and around 5 min boiling at 60°C.
  • Gu*HCl Guanidine hydrochloride in 50mM Tri- ethylammoniumhydrogencarbonat
  • the affinity of the conjugate can be checked using ELISA technology or surface plasmon resonance analysis (BIA-coreTM measurement).
  • the conjugate concentration can be measured by the person skilled in the art using customary methods, for example for antibody conjugates by protein determination, (see also Doronina et al; Nature Biotechnol.2003; 21:778-784 und Poison et al, Blood 2007; 1102:616- 623).
  • TPP-9574 are antibodies comprising one or more of the CDR sequences given in the table above (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) the variable region of the heavy chain (VH) or the variable region of the light chain (VL).
  • the antibodies comprise the indicated variable region of the heavy chain (VH) and/or the indicated variable region of the light chain (VL).
  • the antibodies comprise the indicated region of the heavy chain (IgG heavy chain) and/or the indicated region of the light chain (IgG light chain).
  • TPP-981 is an anti-EGFR antibody comprising a variable region of the heavy chain
  • VH comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 2, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 3, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 4 and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 6, the variable CDR2 sequence of the light chain (L- CDR2), as shown by SEQ ID NO: 7, and the variable CDR3 sequence of the light chain (L- CDR3), as shown by SEQ ID NO: 8.
  • TPP-1015 is an anti-HER2 antibody comprising a variable region of the heavy chain
  • VH comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 12, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 13, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 14, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 16, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 17, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 18.
  • TPP-2658 is an anti-TWEAKR antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 32, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 33, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 34, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 36, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 37, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 38.
  • VH variable region of the heavy chain
  • H-CDR1 sequence of the heavy chain (H-CDR1) as shown by SEQ ID NO: 32
  • TPP-7007 is an anti-TWEAKR antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 62, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 63, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 64, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 66, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 67, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 68.
  • VH variable region of the heavy chain
  • H-CDR1 variable CDR1 sequence of the heavy chain
  • SEQ ID NO: 62 the variable CDR2
  • TPP-2090 is an anti-TWEAKR antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 72, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 73, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 74, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 76, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 77, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 78.
  • VH variable region of the heavy chain
  • H-CDR1 variable CDR1 sequence of the heavy chain
  • SEQ ID NO: 72 the variable CDR2 sequence of
  • TPP-170 is an anti-mesothelin antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 82, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 83, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 84, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 86, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 87, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 88.
  • VH variable region of the heavy chain
  • H-CDR1 variable CDR1 sequence of the heavy chain
  • SEQ ID NO: 82 the variable CDR
  • TPP-9476 is an anti-CD123 antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 92, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 93, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 94, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 96, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 97, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 98.
  • VH variable region of the heavy chain
  • H-CDR1 variable CDR1 sequence of the heavy chain
  • H-CDR1 variable CDR1 sequence of the heavy
  • TPP-9574 is an anti-CXCR 5 antibody comprising a variable region of the heavy chain (VH) comprising the variable CDR1 sequence of the heavy chain (H-CDR1), as shown by SEQ ID NO: 102, the variable CDR2 sequence of the heavy chain (H-CDR2), as shown by SEQ ID NO: 103, and the variable CDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 104, and a variable region of the light chain (VL) comprising the variable CDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 106, the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ ID NO: 107, and the variable CDR3 sequence of the light chain (L-CDR3), as shown by SEQ ID NO: 108.
  • VH variable region of the heavy chain
  • H-CDR1 sequence of the heavy chain (H-CDR1) as shown by SEQ ID NO: 102
  • TPP-981 is an anti-EGFR antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 1 and a variable region of the light chain (VL) represented by SEQ ID NO: 5.
  • TPP-1015 is an anti-HER2 antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 11 and a variable region of the light chain (VL) represented by SEQ ID NO: 15.
  • TPP-2658 is an anti-TWEAKR antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 31 and a variable region of the light chain (VL) represented by SEQ ID NO: 35.
  • TPP-7007 is an anti-TWEAKR antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 61 and a variable region of the light chain (VL) represented by SEQ ID NO: 65.
  • TPP-2090 is an anti-TWEAKR antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 71, and a variable region of the light chain (VL) represented by SEQ ID NO: 75.
  • TPP-170 is an anti-mesothelin antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 81, and a variable region of the light chain (VL) represented by SEQ ID NO: 85.
  • TPP-9476 is an anti-CD123 antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 91, and a variable region of the light chain (VL) represented by SEQ ID NO: 95.
  • TPP-9574 is an anti-CXCR5 antibody preferably comprising a variable region of the heavy chain (VH) represented by SEQ ID NO: 101, and a variable region of the light chain (VL) represented by SEQ ID NO: 105.
  • TPP-981 is an anti-EGFR antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 9 and a region of the light chain represented by SEQ ID NO: 10.
  • TPP-1015 is an anti-HER2 antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 19 and a region of the light chain represented by SEQ ID NO: 20.
  • TPP-2658 is an anti-TWEAKR antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 39 and a region of the light chain represented by SEQ ID NO: 40.
  • TPP-7007 is an anti-TWEAKR antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 69 and a region of the light chain represented by SEQ ID NO: 70.
  • TPP-2090 is an anti-TWEAKR antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 79, and a region of the light chain represented by SEQ ID NO: 80.
  • TPP-170 is an anti-mesothelin antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 89, and a region of the light chain represented by SEQ ID NO: 90.
  • TPP-9476 is an anti-CD123 antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 99, and a region of the light chain represented by SEQ ID NO: 100.
  • TPP-9574 is an anti-CXCR5 antibody preferably comprising a region of the heavy chain represented by SEQ ID NO: 109, and a region of the light chain represented by SEQ ID NO: 110.
  • Composition 0.2 g KCl; 0.2 g KH2PO4 (anhyd); 8.0 g NaCl; 1.15 g Na2HPO4 (anhyd); made up ad I I with H2O Dt doublet of triplets (in NMR) DTT DL-dithiothreitol d. Th. of theory (in chemical yield) EDC N'-(3-dimethylaminopropyl)-N-ethylcarbodi- imide hydrochloride EGFR epidermal growth factor receptor EI electron impact ionization (in MS) ELISA enzyme-linked immunosorbent assay eq.
  • the preparation may comprise a small proportion of the respective other form.
  • the coupling reactions were carried out under argon.
  • the concentrations of the antibodies or ADCs which are indicated in the experimental procedures may differ without a major impact on the outcome.
  • Antibody or ADC concentrations in the range of 1-20 mg/mL can also be used in the coupling reactions, preferred is a concentration range in the coupling reactions of 5-15 mg/mL.
  • the precursor molecules can be added in excesses which may deviate from what is given in the procedures which then may have an impact on the DAR. An excess of 2-20 equivalents of the payload precursor can be used, preferred is the range 4-15 equivalents.
  • the mixture was diluted to a volume of 2.5 ml with PBS buffer which had been adjusted to pH 8 beforehand and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8.
  • the eluate was stirred at RT under argon overnight at pH 8. This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2) to a volume of about 1-5 mL.
  • the mixture was diluted with PBS buffer pH 7.2 to a volume of 2.5 ml and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 7.2 and eluted with PBS buffer pH 7.2. This was followed by concentration by ultracentrifugation and redilution with PBS buffer (pH 7.2) to a volume of about 1-5 mL.
  • the mixture was diluted with PBS Puffer (pH7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was then concentrated by ultracentrifugation to a volume of about 0.300 mL and rediluted with PBS buffer (pH 7.2) to a volume of about 1-5 mL.
  • ADC Precursor Molecules applied to the synthesis of A2DCs [0236] The following payload-linker precursor molecules were employed in the A2DCs and the synthesis has been described in WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578. Novel and advantageous A2DCs can be obtained from specific combinations of payloads, linkers, antibodies, and/or precursor molecules known in the art, according to the formulae and examples disclosed herein.
  • Payload-Linker Intermediates [0266] The synthesis of the payload-linker precursor molecules R1-R6 and Q1-Q9 has been described in WO2016/207089, WO2017/162663, WO2018/114798 and WO2018/114578.
  • A2DC Synthesis Part I [0267] For the synthesis of A2DCs exemplified in the Examples I-1 to I-14 the payload- linker precursor molecules R1-R6 and Q1-Q9 are subsequently coupled to the respective antibodies. The precursor molecules can be combined with each other in different ways for synthesis of A2DCs to addresss the desired biological profiles of the respective A2DCs.
  • PBS Puffer pH7.2
  • the mixture was diluted with PBS Puffer (pH7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was diluted with PBS buffer to a volume of 14 mL, then concentrated by ultracentrifugation to 2 mL, rediluted with PBS buffer (pH 7.2) to a volume of 14 mL and again concentrated to a final volume of 2 mL.
  • the ADC batches obtained with the individual antibodies were characterized as shown in the table below.
  • PBS Puffer pH7.2
  • the mixture was diluted with PBS Puffer (pH7.2) to a volume of 5 mL, applied to 2 PD-10 columns (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer each.
  • the obtained solution was collected and concentrated by ultracentrifugation to give 2 mL, which was rediluted with PBS buffer (pH 7.2) to a volume of 14 mL and which was concentrated again to give a final volume of 3 mL.
  • the ADC batches obtained with the individual antibodies were characterized as shown in the table below.
  • PBS buffer pH7.2
  • the obtained solution was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 500 ⁇ L.
  • This sample was divided and the cystein DAR (C-DAR) was determined, which at the end also represents the C-DAR in the A2DC.250 ⁇ L of the ADC intermediate was used for subsequent lysine coupling: [0278] 250 ⁇ L of the obtained cysteine-linked ADC solution was diluted with PBS buffer to a volume of about 550 ⁇ L. Under an argon atmosphere, 10 Eq (0.31 mg) of Intermediate Q2 dissolved in 50 ⁇ L DMSO was added and the solution was stirred for 2 hours at RT.
  • the mixture was diluted with PBS buffer (pH 7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was then concentrated by ultracentrifugation to a volume of about 100 ⁇ L and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL.
  • the A2DC batch obtained was characterized as shown in the table below.
  • PBS buffer pH7.2
  • the obtained solution was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 500 ⁇ L. This sample was divided and the cystein DAR (C-DAR) was determined, which at the end also represents the C-DAR in the A2DC.250 ⁇ L of the ADC intermediate was used for subsequent lysine coupling: [0285] The solution of 250 ⁇ L of the obtained cysteine-linked ADC was diluted with PBS buffer to a volume of 550 ⁇ L. Under an argon atmosphere, 10 Eq (0.4 mg) of Intermediate R2 dissolved in 50 ⁇ L DMSO was added and the solution was stirred for 2 hours at RT.
  • PBS buffer pH 7.2
  • the mixture was diluted with PBS buffer (pH7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was then concentrated by ultracentrifugation to a volume of about 100 ⁇ L and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL.
  • the A2DC batch obtained was characterized as shown in the table below.
  • PBS buffer pH 7.2
  • the obtained solution was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 500 ⁇ L.
  • This sample was divided and the cystein DAR (C-DAR) was determined, which in the end also represents the C-DAR in the A2DC.250 ⁇ L of the ADC intermediate was used for subsequent lysine coupling: [0289]
  • the solution of 250 ⁇ L of the obtained cysteine-linked ADC was diluted with PBS buffer to a volume of 550 ⁇ L. Under an argon atmosphere, 10 Eq (0.31 mg) of Intermediate Q1 dissolved in 50 ⁇ L DMSO was added and the solution was stirred for 2 hours at RT.
  • the mixture was diluted with PBS buffer (pH 7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was then concentrated by ultracentrifugation to a volume of about 100 ⁇ L and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL.
  • the A2DC batch obtained was characterized as shown in the table below.
  • PBS buffer pH7.2
  • the obtained solution was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 500 ⁇ L.
  • This sample was divided and the cystein DAR (C-DAR) was determined, which in the end also represents the C-DAR in the A2DC.250 ⁇ L of the ADC intermediate was used for subsequent lysine coupling: [0291]
  • the solution of 250 ⁇ L of the obtained cysteine-linked ADC was diluted with PBS buffer to a volume of 550 ⁇ L. Under an argon atmosphere, 10 Eq (0.39 mg) of Intermediate R 5 dissolved in 50 ⁇ L DMSO was added and the solution was stirred for 2 hours at RT.
  • the mixture was diluted with PBS Puffer (pH7.2) to a volume of 2.5 mL, applied to a PD-10 column (Sephadex® G-25 GE Healthcare) and eluted with 3.5 mL PBS buffer.
  • the obtained solution was then concentrated by ultracentrifugation to a volume of about 100 ⁇ L and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL.
  • the A2DC batch obtained was characterized as shown in the table below.
  • Biological characterization CTG Assay [0292] The cells were cultivated according to the standard method using the growth media listed under C-I.
  • the test was carried out by detaching the cells with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleting, resuspending in culture medium, counting and sowing into a 96-well culture plate with white bottom (Costar #3610) (at 75 pl/well, the following cell numbers are per well: NCI-H292: 2500 cells/well, BxPC32500 cells/well, LoVo 3000 cells/well) and incubating in an incubator at 37° C. and 5% carbon dioxide.
  • the antibody drug conjugates were added in 25 pi of culture medium (concentrated four-fold) to the cells to give final antibody drug conjugate concentrations of 3x10- 7 M to 3xlO-11 M on the cells (triplicates).
  • the cells were then incubated in an incubator at 37° C. and 5% carbon dioxide.
  • the cell activity at the start of the drug treatment was determined using the Cell Titer Glow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571).
  • the plates were then covered with aluminium foil, shaken on the plate shaker at 180 rpm for 2 minutes, allowed to stand on the laboratory bench for 8 minutes and then measured using a luminometer (Victor X2, Perkin Elmer).
  • the substrate detects the ATP content of the living cells generating a luminescence signal whose intensity is directly proportional to the viability of the cells. After 72 h of incubation with the antibody drug conjugates, the viability of these cells was then also determined using the Cell Titer Glow luminescent cell viability assay as described above.
  • the IC50 of the growth inhibition was calculated in comparison to day 0 using the DRC (Dose Response Curve) analysis spreadsheets and a 4-parameter fit.
  • the DRC analysis spreadsheet is a biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).
  • IDBS ID Business Solutions Ltd., Guildford, UK.
  • Table 2 and Table 3 below sets out the IC50 values for representative working examples from this assay: Table 2: TWEAKR-A2DCs
  • the activity data reported relate to the working examples described in the present experimental section, with the drug/mAB ratios indicated. The values may possibly deviate for different drug/mAB ratios.
  • the IC50 values are means of several independent experiments or individual values.
  • the action of the antibody drug conjugates was selective for the respective isotype control comprising the respective linker and toxophore.
  • Table 3 CXCR 5 -A2DCs MTT Assay [0295] The cells were cultivated according to the standard method using the growth media listed under C-I. The test was carried out by detaching the cells with a solution of Accutase in PBS buffer (from Biochrom AG #L2143). The cells were pelletized and resuspended in culture medium.
  • a defined cell number was sowed into a 96-well culture plate with white bottom (from Costar #3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well) in a total volume 100 ⁇ L. Then the cells were incubated in an incubator at 37° C. and 5 % carbon dioxide for48 h, After this incubation the medium was exchanged by fresh medium. Afterwards the antibody drug conjugates were added in a concentration range from I0-5 M to I0-13 Madded to the cells (in triplicates) in a final volume of 10 ⁇ L followed by an incubation at 37° C. and 5 % CO2.
  • the cell proliferation was detected using the MTT assay (ATCC, Manassas, Va., USA, catalogue No.30-1010K). To this end, the MTT reagent was added to the cells followed by a 4 h incubation The lysis of the cells occurred overnight by addition of the detergent. The dye formation was detected at 570 nm (Infinite M1000 pro, Tecan). The measured data were used to calculate the IC50 of the growth inhibition using the DRC (dose response curve). The proliferation of cells which were not treated with test substance but were otherwise identically treated was defined as the 100% value. [0296] The activity data reported relate to the working examples described in the present experimental section, with the drug/mAB ratios indicated.
  • the values may possibly deviate for different drug/mAB ratios.
  • the IC50 values are means of several independent experiments or individual values.
  • the action of the antibody drug conjugates was selective for the respective isotype control comprising the respective linker and toxophore. Results are summarized in the following tables (4, 5, 6, 7).
  • Table 4 Summary of mean IC50 values of Mesothelin-A2DC Table 5: Summary of mean IC50 values of EGFR-A2DCs Table 6: Summary of mean IC50 values of HER2-A2DCs Table 7: Summary of mean IC50 value of CD123-A2DCs Internalization Assay [0297] Internalization is a key process which enables specific and efficient provision of the cytotoxic payload in antigen-expressing cancer cells via antibody drug conjugates (ADC). This process is monitored via fluorescent labelling of specific antibodies and an isotype control antibody. First, the fluorescent dye was conjugated to lysines of the antibody.
  • ADC antibody drug conjugates
  • Conjugation was carried out using a two-fold molar excess of CypFier 5E mono NFiS ester (Batch 357392, GE Fiealthcare) at pFi 8.3.
  • the reaction mixture was purified by gel chromatography (Zeba Spin Desalting Columns, 40K, Thermo Scientific, No.87768; elution buffer: DULBECCO’S PBS, Sigma-Aldrich, No. D8537), to eliminate excess dye and to adjust the pH.
  • the protein solution was concentrated using VIVASPIN 500 columns (Sartorius stedim biotec).
  • the dye load of the antibodies examined here and the isotype control were of a comparable order of magnitude. In cell binding assays, it was confirmed that the coupling did not lead to any change in the affinity of the antibodies.
  • the labelled antibodies were used for the internalization assay. Prior to the start of the treatment, cells (2x104/well) were sown in 100 ⁇ li medium in a 96-well MTP (fat, black, clear bottom No 4308776, from Applied Biosystems).
  • a tumour grows. Treatment is commenced after the tumour is established, at a tumour size of approximately 40 mm2. To examine the effect on laiger tumours, treatment may be initiated at a tumour size of 50-100 mm2.
  • Treatment with A2DCs is carried out intraperitoneally or via the intravenous (i.v.) route into the tail vein of the mouse. The A2DC is administered in a volume of 5 ml/kg.
  • the treatment protocol depends on the pharmacokinetics of the antibody. With the conjugates according to the invention, treatment is effected once or twice per week for 2 or 3 weeks as the standard. For highly efficacious compounds, a protocol with a single treatment may be employed.
  • the treatment may also be continued, or a second cycle of three treatment days may follow at a later time.
  • 8 animals are used per treatment group.
  • one group is treated as control group only with the buffer, according to the same protocol.
  • the tumour area is measured regularly in two dimensions (length/width) using a caliper.
  • the tumour area is determined as length x width.
  • the ratio of the mean tumour area of the treatment group to that of the control group is stated as T/Carea.
  • the A2DC example I-1k-7007* binding to the TWEAK-receptor was tested at a dose of 5 mg/kg in mice (NMRI, female) bearing NCI-H292 tumors which highly express the antigen.
  • example I-1k-7007* was compared to an isotype-conjugate as well as the vehicle (PBS). Strong efficacy of tumor inhbition of example I-1k-7007* starting early after the first dosing was observed which lasted for approximately 40 days before tumor re-growth occurred. Compared against the vehicle the isotype-conjugate demonstrated slight efficacy only.
  • FIG.3 [0304] In addition to this xenograft model also the syngeneic (mouse) tumor models CT26 and MC38 were investigated, both expressing the TWEAK-receptor as confirmed by immunohisto-chemistry.
  • the A2DC example I 11k-2658* was intraperitoneally injected in CT26 tumor bearing mice (Balb/c, female) as a single treatment at two different doses (2.5 and 5 mg/kg) and tumor growth was observed for approximately 3 weeks. As result a strong and dose-dependent tumor growth inhibition by example I-11k-2658* could be demonstrated. A similar strong efficacy of 11k-2658* was found in MC38-tumor bearing mice treated with 5 mg/kg for three times. (FIG.4) [0305] In all studies the A2DC compounds were tolerated well without affecting body weight or behaviour of the rodents. There were no suspicious findings in macroscopic necropsies.
  • the antibodies for example TPP-2658, TPP-2090, TPP-7007, TPP-1015, TPP-170, TPP-6013, TPP-9476, TPP-9574 and TPP-981, were produced in transient mammalian cell cultures as described by Tom et ah, Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007.
  • the antibodies for example TPP-2658, TPP-2090, TPP-7007, TPP-1015, TPP-170, TPP-6013, TPP-9476, TPP-9574 and TPP-981, were obtained from the cell culture supernatants.
  • the cell supernatants were clarified by centrifugation of cells.
  • the cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column.
  • the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with about 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride.
  • the antibodies were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.
  • the commercially available antibodies were purified from the commercial products by standard chromatography methods (protein A chromatography, preparative gel filtration chromatography (SEC—size exclusion chromatography)).
  • free cysteine residues can be site-specifically engineered into an antibody.
  • the coupling reactions with the electrophiles described in the section ‘ADC precursor molecules‘ were usually carried out under argon as described in the general procedures in the section ‘A2DC examples‘.
  • concentrations of the final A2DC samples were optionally adjusted to the range of 0.5-15 mg/ml by redilution.
  • the respective protein concentrations, stated in the working examples, of the A2DC solutions were determined.
  • antibody loading drug/mAb ratio
  • the A2DCs shown in the examples may also be present to a lesser or higher degree in the form of the hydrolysed open-chain succinamides attached to the antibodies.
  • the KSP-I-A2DCs attached through the linker substructure to thiol groups of the antibodies may optionally also be hydrolized after the coupling by rebuffering and stirring at pH 8 for about 20-24 h to obtain A2DCs attached via open-chain succinamides.
  • #1 represents the sulphur bridge to the antibody, and #2 the point of attachment to the modified KSP inhibitor.
  • Such A2DCs where the linker is attached to the antibodies through hydrolysed open- chain succinamides show higher stability towards de-conjugation by retro-Michael reaction and can be prepared as shown in the scheme below and described in General Procedure A (Lerchen et al., Angew. Chem. Int.
  • AK 1 can mean: Examples a: Anti-EGFR mAb (Cetuximab) (TPP981), partially reduced Examples e: Anti-Her2 mAb (TPP 1 015), partially reduced Examples k-7007: Anti-TWEAKR mAb (TPP-7007), partially reduced Examples k-2658: Anti-TWEAKR mAb (TPP-2658), partially reduced Examples k-2090: Anti-TWEAKR mAb (TPP-2090), partially reduced Examples c: Anti-CD123 mAb (TPP-9476), partially reduced Examples x: Anti-CXCR 5 mAb (TPP-9574), partially reduced where ⁇
  • A2DCs and A2DCs have been characterized by mass spectrometric analysis.
  • Molecular weight analysis was performed using a combination of HPLC and ESI-Q- TOF consisting of e.g. an I-class HPLC (Waters) for sample desalting and separation and an Impact HD mass spectrometer (Bruker Daltonik, Bremen) equipped with instrument control and acquisition software HyStar 3.2, ESI Compass 1.7 and Maximum Entropy Deconvolution Option for MS analysis.
  • Chromatographic system and conditions column: Acquity UPLC BEH300C4 1.7 ⁇ m, 1.0x50mm; column temperature: 70°C; flow: 200 ⁇ l/min; mobile phase solution A: 0.1% formic acid, 94,9% water, 5% ACN; mobile phase solution B: 0.1% formic acid, 9.9% water, 10% ACN and 80% 2-propanol.
  • Binary gradient profile for complete antibody 2min 5% B, 2,5min 50% B, 3,5min 50% B, 5min 95%B, 5,1min 5% B, 5,6min 95% B, 5,7min 5% B, 6,2min 95% B, 6,3min 5% B, 7,5min 5% B
  • Binary gradient profile for reduced antibody 2min 5% B, 4min 30% B, 5min 50% B, 7,5min 50%B, 8,5min 95% B, 8,6min 5% B, 9,1min 95% B, 9,3min 5% B, 9,8min 95% B, 9,9min 5% B, 12min 5% B
  • Cys-coupled A2DCs determination of the molecular weights of the individual conjugate species were measured after deglycosylation and reduction.
  • the cooled sample was acidified with 2 ⁇ l of 10% formic acid/water and then analyzed by mass spectrometry as described above. For DAR determination, all spectra over the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution of light and heavy chain.
  • Average loading of the antibody with toxophors was calculated from the peak areas determined by integration as double the sum of the HC-Load and the LC-load, whereas the HC- load is the sum of toxophor number weighted integration results of all heavy chain (HC) -peaks divided by the sum of the singly weighted integration results of the HC- peaks and whereas the LC-load is the sum of toxophor number weighted integration results of the light chain (LC)-peaks divided by the sum of the singly weighted integration results of all LC peaks. [0332] For Lys-coupled A2DCs determination of the molecular weights of the individual conjugate species were measured after deglycosylation.
  • the conjugate concentration can be measured by the person skilled in the art using customary methods, for example for antibody conjugates by protein determination, (see also Doronina et al; Nature Biotechnol.2003; 21:778-784 und Poison et al, Blood 2007; 1102:616- 623).
  • Method 1 Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T31.8 ⁇ 50 x 1 mm; mobile phase A: 1 L of water + 0.25 mL of 99% strength formic acid; mobile phase B: 1 L of acetonitrile + 0.25 mL of 99% strength formic acid; gradient: 0.0 min 90% A ⁇ 1.2 min 5% A ⁇ 2.0 min 5% A oven: 50°C; flow rate: 0.40 mL/min; UV detection: 208 – 400 nm.
  • Method 2 System MS: Thermo Scientific FT-MS; System UHPLC+: Thermo Scientific UltiMate 3000; Column: Waters, HSST3, 2.1 x 75 mm, C181.8 ⁇ m; Eluent A: 1 l Water + 0.01% Formic acid; Eluent B: 1 l Acetonitrile + 0.01% Formic acid; Gradient: 0.0 min 10% B ⁇ 2.5 min 95% B ⁇ 3.5 min 95% B; Oven: 50°C; Flow: 0.90 ml/min; UV- Detection: 210 nm/ Optimum Integration Path 210-300 nm [0336] Method 3 (LC-MS): Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T31.8 ⁇ m 50 x 1 mm; Eluent A: 1 l Water + 0.25 ml 99%ige Formic acid , Eluent B: 1
  • Method 4 System MS: ThermoFisherScientific LTQ-Orbitrap-XL; System HPLC: Agilent 1200SL; Column: Agilent, POROSHELL 120, 3 x 150 mm, SB – C182.7 ⁇ m; Eluent A: 1 l water + 0.1% trifluoroacetic acid; Eluent B: 1 l Acetonitrile + 0.1% trifluoroacetic acid; Gradient: 0.0 min 2% B ⁇ 0.3 min 2% B ⁇ 5.0 min 95% B ⁇ 10.0 min 95% B; Oven: 40°C; Flow: 0.75 ml/min; UV-Detection: 210 nm [0338] Method 5 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters, HSST3, 2.1 x 50 mm, C181.8 ⁇ m; Eluent A: 1 l Water + 0.0
  • N,N-diisopropylethylamine (1.9 ml, 11 mmol; CAS-RN:[7087-68-5]) was slowly added to a solution of (2S)-4-(benzyloxy)-2- ⁇ [(2S)-2- ⁇ [(benzyloxy)carbonyl]amino ⁇ propanoyl]amino ⁇ -4-oxobutanoic acid (988 mg, 2.21 mmol), tert- butyl L-asparaginate (511 mg, 2.65 mmol) and (1.01 g, 2.65 mmol; CAS-RN:148893-10-1) in DMF (10 ml) and the reaction was stirred at room temperature for 5 min.
  • Zinc chloride (14.0 mg, 102 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 1 hour, zinc chloride (14.0 mg, 102 ⁇ mol) was added, and the reaction was stirred at 50 °C for 1 hour. Zinc chloride (14.0 mg, 102 ⁇ mol) was again added, and the reaction was stirred at 50 °C for another hour Ethylene diamine-N,N,N',N'-tetraacetic acid (29.9 mg, 102 ⁇ mol; CAS-RN:[60-00-4]) was added followed by 2ml water/0.1%TFA. The mixture was filtered and the filtrate was purified by preparative HPLC yielding 15 mg of the product.
  • the reaction mixture was stirred at room temperature for 2 hours.
  • the reaction was quenched with water, and the mixture was extracted with ethyl acetate.
  • the organic phase was dried over sodium sulfate, filtered and evaporated to dryness.
  • the crude was purified by column chromatography with dichloromethane/ methanol (20:1/v:v) to yield 5.0 g of the product.
  • N-(tert-butoxycarbonyl)-L-glutamic acid (1.55 g, 6.25 mmol; CAS-RN:2419-94-5), was dissolved in N,N-dimethylformamide (50 ml), then benzyl N-(15-amino-4,7,10,13- tetraoxapentadecanan-1-oyl)-L-valyl-L-alaninate (8.00 g, 12.5 mmol), (4.76 g, 12.5 mmol; CAS-RN:148893-10-1) and ethyldiisopropylamine (2.4 g, 18.8 mmol) were added. The reaction mixture was stirred at room temperature for 2 h.
  • Zinc chloride (70.1 mg, 515 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 1 hour, zinc chloride (70.1 mg, 515 ⁇ mol) was added and the reaction was stirred at 50 °C. After one hour more zinc chloride (70.1 mg, 515 ⁇ mol) was added and the reaction was stirred at 50 °C for 1 hour.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid 150 mg, 515 ⁇ mol; CAS-RN:[60-00-4] was added and the mixture was stirred for a few minutes. The reaction was filtered and the solids were washed with acetonitrile.
  • Zinc chloride (108 mg, 792 ⁇ mol) was added, and the reaction was stirred at 50 °C for 4 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (231 mg, 792 ⁇ mol; CAS-RN:[60-00-4]) was added followed by 2 ml water/0.1%TFA. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 72 mg of the product.
  • Zinc chloride (40.6 mg, 298 ⁇ mol) was added, and the reaction was stirred at 50 °C for 8 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (87.1 mg, 298 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was diluted with water/acetonitrile. The reaction was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 54 mg of the product.
  • Zinc chloride (258 mg, 1.89 mmol) was added, and the reaction was stirred at 50 °C for 3 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (554 mg, 1.89 mmol; CAS-RN:60-00-4) was added followed by 5 ml water/0.1%TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 284 mg of the product.
  • Zinc chloride (12.7 mg, 93.0 ⁇ mol) was added, and the reaction was stirred at 50 °C for 1 hour.
  • Ethylene diamine-N,N,N',N'- tetraacetic acid (27.2 mg, 93.0 ⁇ mol; CAS-RN:60-00-4) was added followed by 4 ml water/acetonitrile (1:1) and TFA (20 ⁇ l) and the mixture was stirred for 10 minutes. The resulting solids were filtered off and the mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 17 mg of the product.
  • Zinc chloride (62.8 mg, 461 ⁇ mol) was added, and the reaction was stirred at 50°C for 1 hour.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (135 mg, 461 ⁇ mol; CAS-RN:[60-00-4]) was added followed by 2 ml water/0.1%TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 69 mg of the product.
  • Zinc chloride (14.8 mg, 109 ⁇ mol) was added, and the reaction was stirred at 50 °C for 2 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid 31.7 mg, 109 ⁇ mol; CAS-RN:60-00-4) was added followed by 2 ml water/0.1%TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 25 mg of the product.
  • the mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour.
  • the solids were filtered off and the filtrate was concentrated under reduced pressure.
  • the remaining residue was dissolved in acetonitrile/water and was lyophilized to give 20 mg of the title product.
  • the mixture was hydrogenated at room temperature and under standard hydrogen pressure for 1 hour.
  • the solids were filtered off and the filtrate was concentrated under reduced pressure.
  • the remaining residue was dissolved in acetonitrile/water and was lyophilized to give 10 mg of the title product.
  • Zinc chloride (6.54 mg, 48.0 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 3 hours zinc chloride (6.54 mg, 48.0 ⁇ mol) was added and the reaction was stirred at 50 °C for 1 hour.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (28.0 mg, 95.9 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was stirred for 10 min. Water/0.1%TFA was added and the solids were filtered off. The filtrate was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 34 mg of the product.
  • Zinc chloride (3.17 mg, 23.2 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 1.5 hours, zinc chloride (3.17 mg, 23.2 ⁇ mol l) was added and the raction was stirred at 50 °C for 1.5 hours. Zinc chloride (3.17 mg, 23.2 ⁇ mol) was added again and the reaction was stirred at 50°C until completion. Ethylene diamine-N,N,N',N'- tetraacetic acid (20.4 mg, 69.7 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was stirred for 10 min. Water/0.1%TFA was added and the solids were filtered off.
  • Zinc chloride (1.06 mg, 7.78 ⁇ mol) was added, and the reaction was stirred at 50 °C. After 1 hour, more zinc chloride (1.06 mg, 7.78 ⁇ mol) was added, and the reaction was stirred at 50 °C for another hour. This procedure was repeated 4 times until the reaction was completed.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (2.27 mg, 7.78 ⁇ mol; CAS-RN:60-00-4) was added, followed by water/0.1%TFA and the mixture was stirred for a few minutes. The mixture was filtered and the filtrate was purified by preparative HPLC yielding 2.2 mg of the product.
  • the mixture was hydrogenated at room temperature and under standard hydrogen pressure for 12 hour.
  • the solids were filtered off and the residue was washed with methanol/DCM.
  • the filtrate was concentrated under reduced pressure.
  • the remaining residue was dissolved in acetonitrile/water and was lyophilized to give 97 mg of the title product.
  • Zinc chloride (28.6 mg, 210 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 2 hours, zinc chloride (28.6 mg, 210 ⁇ mol) was added, and the reaction was stirred at 50 °C for 2 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (61.2 mg, 210 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was stirred for 10 minutes followed by the addition of water/TFA (0.1%). The mixture was filtered and the filtrate was purified by preparative HPLC yielding 41 mg of the product.
  • Zinc chloride (4.71 mg, 34.5 ⁇ mol) was added, and the reaction was stirred at 50 °C for 2.5 hours.
  • Ethylene diamine-N,N,N',N'- tetraacetic acid (10.1 mg, 34.5 ⁇ mol; CAS-RN:60-00-4) was added, followed by acetonoitrile and water. The mixture was filtered and the filtrate was purified by preparative HPLC yielding 11 mg of the product.
  • Zinc chloride (5.04 mg, 37.0 ⁇ mol) was added, and the reaction was stirred at 50 °C for 2 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (10.8 mg, 37.0 ⁇ mol; CAS-RN:[60-00-4]) was added, followed by 2ml water/0.1%TFA. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC yielding 12mg of the product.
  • Zinc chloride (9 mg, 67.1 ⁇ mol) was added, and the reaction was stirred at 50 °C. After stirring for 1 hour, zinc chloride (9 mg, 67.1 ⁇ mol) was added and the reaction was stirred at 50 °C for another hour. To drive the reaction to completion, more zinc chloride (9 mg, 67.1 ⁇ mol) was added and the reaction was stirred at 50 °C for 1 hour. Ethylene diamine-N,N,N',N'-tetraacetic acid (19.6 mg, 67.1 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was stirred for a few minutes followed by the addition of water/0.1%TFA.
  • Zinc chloride (10.9 mg, 79.9 pmol) was added, and the reaction was stirred at 50 °C for 1 hours. More zinc chloride (10.9 mg, 79.9 ⁇ mol) was added and the reaction was stirred at 50 °C for 1 hour, this step was repeated until the reaction was completed.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (23.4 mg, 79.9 ⁇ mol; CAS-RN:[60-00-4]) was added and the mixture was stirred for a few minutes followed by the addition of water/0.1%TFA. The mixture was filtered and the filtrate was purified by preparative HPLC yielding 15 mg of the product.
  • the concentration of the antibody solution can also vary from the standard procedure in a range between 1 mg/ml and 30 mg/ml).
  • the mixture was stirred at RT for 30 min, and then an excess of 2-20 equivalents, preferably 2-10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC precursor molecule) dissolved in 0.05 mL DMSO was added. After stirring at RT for a further 90 min, the mixture was diluted to a volume of 2.5 ml with PBS buffer which had been adjusted to pH 8 beforehand and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8.
  • PBS buffer which had been adjusted to pH 8 beforehand and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8.
  • the concentration of the antibody solution can also vary from the standard procedure in a range between 1 mg/ml and 30 mg/ml).
  • the mixture was stirred at RT for 30 min, and then an excess of 2-20 equivalents, preferably 2-10 equivalents, typically 7 equivalents (0.000233 mmol) of the respective intermediate (ADC precursor molecule) dissolved in 0.05 mL of DMSO was added. After stirring at RT for a further 90 min, the mixture was diluted with PBS buffer pH 7.2 to a volume of 2.5 ml and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 7.2 and eluted with PBS buffer pH 7.2.
  • PD 10 column Sephadex® G-25, GE Healthcare
  • the mixture was stirred at RT for 30 min, and then an excess of 2-10 equivalents, typically 7 equivalents (0.000933 mmol) of the respective intermediate (ADC precursor molecule) dissolved in 0.2 mL of DMSO was added. After stirring at RT for a further 90 min, the mixture was diluted with PBS buffer pH 7.2 to a volume of 2.5 ml and then passed through a PD 10 column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH 7.2 and eluted with PBS buffer pH 7.2.
  • PD 10 column Sephadex® G-25, GE Healthcare
  • Example II-1 [0723] ADCs were synthesized by coupling of Intermediate 158 to different antibodies using one of the general procedures described above as indicated in the table.
  • Example II-2 [0724] ADCs were synthesized by coupling of Intermediate 159 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 159 to different antibodies were characterized as shown in the table below:
  • Example II-3 [0725] ADCs were synthesized by coupling of Intermediate 160 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 160 to different antibodies were characterized as shown in the table below:
  • Example II-4 [0726] ADCs were synthesized by coupling of Intermediate 161 to different antibodies using one of the general procedures described above as indicated in the table.
  • Example II-5 ADCs were synthesized by coupling of Intermediate 162 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 162 to different antibodies were characterized as shown in the table below:
  • Example II-6 ADCs were synthesized by coupling of Intermediate 163 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 163 to different antibodies were characterized as shown in the table below:
  • Example II-6a Metabolites 1 [0729] The synthesis of this isomeric non-permeable metabolites 1 released from the A2DC in example 6 has been described in WO2018/114804.
  • Example II-6b Metabolite 2 [0730] The synthesis of this permeable metabolite 2 released from the A2DC in example 6 was synthesized according to the following procedure.
  • Step1 To a solution of (2S)-4-[ ⁇ (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol- 2-yl]-2,2-dimethylpropyl ⁇ (hydroxyacetyl)amino]-2-( ⁇ [2- (trimethylsilyl)ethoxy]carbonyl ⁇ amino)butanoic acid (40.0 mg, 60.8 ⁇ mol) in DMF (7 ml) was added 3-amino-N-methylpropanamide*HCl (10.1 mg, 73.0 ⁇ mol), HATU (27.7 mg, 73.0 ⁇ mol; CAS-RN:148893-10-1) and N,N-diisopropylethylamine (32 ⁇ l, 182 ⁇ mol; CAS-RN:7087-68-5] and the reaction was stirred at room temperature for 1 h.
  • Zinc chloride (41.3 mg, 303 ⁇ mol) was added, and the reaction was stirred at 50 °C for 2.5 hours.
  • Ethylene diamine-N,N,N',N'-tetraacetic acid (88.6 mg, 303 ⁇ mol; CAS-RN:[60-00-4]) was added followed by acetonitrile and water and the mixture was stirred for a few minutes.
  • Example II-7 ADCs were synthesized by coupling of Intermediate 164 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 164 to different antibodies were characterized as shown in the table below:
  • Example II-8 ADCs were synthesized by coupling of Intermediate 165 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 165 to different antibodies were characterized as shown in the table below:
  • Example II-9 ADCs were synthesized by coupling of Intermediate 166 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 166 to different antibodies were characterized as shown in the table below:
  • Example II-10 ADCs were synthesized by coupling of Intermediate 167 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 167 to different antibodies were characterized as shown in the table below:
  • Example II-11 ADCs were synthesized by coupling of Intermediate 169 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 169 to different antibodies were characterized as shown in the table below:
  • Example II-12 ADCs were synthesized by coupling of Intermediate 169 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 169 to different antibodies were characterized as shown in the table below:
  • Example II-13 [0741] ADCs were synthesized by coupling of Intermediate 170 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 170 to different antibodies were characterized as shown in the table below:
  • Example II-14 ADCs were synthesized by coupling of Intermediate 171 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 171 to different antibodies were characterized as shown in the table below:
  • Example II-15 ADCs were synthesized by coupling of Intermediate 172 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 172 to different antibodies were characterized as shown in the table below:
  • Example II-16 ADCs were synthesized by coupling of Intermediate 173 to different antibodies using one of the general procedures described above as indicated in the table.
  • Example II-17 [0745] ADCs were synthesized by coupling of Intermediate 174 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 174 to different antibodies were characterized as shown in the table below:
  • Example II-18 ADCs were synthesized by coupling of Intermediate 175 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 175 to different antibodies were characterized as shown in the table below:
  • Example II-19 ADCs were synthesized by coupling of Intermediate 176 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 176 to different antibodies were characterized as shown in the table below:
  • Example II-20 [0748] ADCs were synthesized by coupling of Intermediate 178 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 178 to different antibodies were characterized as shown in the table below: Example II-21 [0749] ADCs were synthesized by coupling of Intermediate 178 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 178 to different antibodies were characterized as shown in the table below: Example II-22 [0750] ADCs were synthesized by coupling of Intermediate 179 to different antibodies using one of the general procedures described above as indicated in the table.
  • Example II-23 [0751] ADCs were synthesized by coupling of Intermediate 180 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 180 to different antibodies were characterized as shown in the table below: Example II-24
  • ADCs were synthesized by coupling of Intermediate 181 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 181 to different antibodies were characterized as shown in the table below:
  • Example II-25 [0753] ADCs were synthesized by coupling of Intermediate 182 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 182 to different antibodies were characterized as shown in the table below: Example II-26
  • ADCs were synthesized by coupling of Intermediate 183 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 183 to different antibodies were characterized as shown in the table below:
  • ADCs were synthesized by coupling of Intermediate 184 to different antibodies using one of the general procedures described above as indicated in the table.
  • the ADC batches obtained after coupling of Intermediate 184 to different antibodies were characterized as shown in the table below:
  • Example II-28 ADCs were synthesized by coupling of Intermediate 185 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 185 to different antibodies were characterized as shown in the table below: Example II-29 [0757] ADCs were synthesized by coupling of Intermediate 186 to different antibodies using one of the general procedures described above as indicated in the table. The ADC batches obtained after coupling of Intermediate 186 to different antibodies were characterized as shown in the table below: Biological characterization CTG Assay [0758] The cells were cultivated according to the standard method using the growth media listed under C-I.
  • the test was carried out by detaching the cells with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelleting, resuspending in culture medium, counting and sowing into a 96-well culture plate with white bottom (Costar #3610) (at 75 pl/well, the following cell numbers are per well: NCI-H292: 2500 cells/well, BxPC32500 cells/well, LoVo 3000 cells/well) and incubating in an incubator at 37° C. and 5% carbon dioxide.
  • the antibody drug conjugates were added in 25 pi of culture medium (concentrated four-fold) to the cells to give final antibody drug conjugate concentrations of 3xl0- 7 M to 3xlO-11 M on the cells (triplicates).
  • the cells were then incubated in an incubator at 37° C. and 5% carbon dioxide.
  • the cell activity at the start of the drug treatment was determined using the Cell Titer Glow (CTG) luminescent cell viability assay (Promega #G7573 and #G7571).
  • the plates were then covered with aluminium foil, shaken on the plate shaker at 180 rpm for 2 minutes, allowed to stand on the laboratory bench for 8 minutes and then measured using a luminometer (Victor X2, Perkin Elmer).
  • the substrate detects the ATP content of the living cells generating a luminescence signal whose intensity is directly proportional to the viability of the cells. After 72 h of incubation with the antibody drug conjugates, the viability of these cells was then also determined using the Cell Titer Glow luminescent cell viability assay as described above.
  • the IC50 of the growth inhibition was calculated in comparison to day 0 using the DRC (Dose Response Curve) analysis spreadsheets and a 4-parameter fit.
  • the DRC analysis spreadsheet is a biobook spreadsheet developed by Bayer Pharma AG and Bayer Business Services on the IDBS E-WorkBook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK).
  • IDBS ID Business Solutions Ltd., Guildford, UK.
  • the activity data reported relate to the working examples described in the present experimental section, with the drug/mAB ratios indicated. The values may possibly deviate for different drug/mAB ratios.
  • the IC50 values are means of several independent experiments or individual values.
  • the action of the antibody drug conjugates was selective for the respective isotype control comprising the respective linker and toxophore.
  • Table 9a-f sets out the IC50 values for representative working examples from this assay: Table 9a:TWEAKR-A2DCs
  • Table 9b Mesothelin-A2DCs
  • Table 9c EGFR-A2DCs
  • Table 9d HER2-ADCs
  • Table 9e CXCR5-A2DCs
  • Table 9f CD123-A2DCs MTT Assay
  • the cells were cultivated according to the standard method using the growth media listed under C-I. The test was carried out by detaching the cells with a solution of Accutase in PBS (from Biochrom AG #L2143), pelletizing, resuspending in culture medium, counting and sowing into a 96-well culture plate with white bottom (from Costar #3610) (NCI H292: 2500 cells/well; SK-HEP-1: 1000 cells/ well; KPL-4: 1200 cells/well; in total volume 100 pi). The cells were then incubated in an incubator at 37° C. and 5% carbon dioxide. After 48 h, the medium was replaced.
  • the antibody drug conjugates in 10 pi of culture medium in concentrations from IO-5M to IO-13M were then pipetted onto the cells (in triplicate), and the assay was then incubated in an incubator at 37° C. and 5% carbon dioxide. After 96 h, cell proliferation was detected using the MTT assay (ATCC, Manassas, Va., USA, catalogue No.30- 1010K). To this end, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight by addition of the detergent. The dye formed was detected at 570 nm (Infinite MlOOO pro, Tecan). The measured data were used to calculate the IC50 of the growth inhibition using the DRC (dose response curve).
  • Table 11 Specified example 11m-170: A2DC retains by-stander killing of permable ADC component: Potency of A2DC at Mesothelin-transfected HT29 cells, HT29 wild type and co-culture in comparison to isotype control A2DC and the respective ADC components in the A2DC Table 12: Specified example 15m-170: Potency of A2DC at Mesothelin-transfected HT29 cells is higher than repective ADC and also dependent on target expression.
  • Internalization Assay [0764] Internalization is a key process which enables specific and efficient provision of the cytotoxic payload in antigen-expressing cancer cells via antibody drug conjugates (ADC). This process is monitored via fluorescent labelling of specific antibodies and an isotype control antibody.
  • the fluorescent dye was conjugated to lysines of the antibody. Conjugation was carried out using a two-fold molar excess of CypFier 5E mono NFiS ester (Batch 357392, GE Healthcare) at pFi 8.3. After the coupling, the reaction mixture was purified by gel chromatography (Zeba Spin Desalting Columns, 40K, Thermo Scientific, No.87768; elution buffer: DULBECCO’S PBS, Sigma-Aldrich, No. D8537), to eliminate excess dye and to adjust the pH. The protein solution was concentrated using VIVASPIN 500 columns (Sartorius stedim biotec).
  • the dye load of the antibodies examined here and the isotype control were of a comparable order of magnitude. In cell binding assays, it was confirmed that the coupling did not lead to any change in the affinity of the antibodies.
  • the labelled antibodies were used for the internalization assay. Prior to the start of the treatment, cells (2x104/well) were sown in 100 pi medium in a 96-well MTP (fat, black, clear bottom No 4308776, from Applied Biosystems).
  • A2DC metabolite formation from A2DCs [0768] For quantification of the A2DC metabolites released from A2DCs, cancer cells were incubated with A2DCs from example 6 for 72h. Cell lysates and corresponding supernatants were collected at various time points and the active payload metabolite was quantified by LC- MS. For calibration, the cell homogenate (for cell lysate) or cell culture medium (for supernatant), was spiked with 0.6–1000 ⁇ g/L of active metabolite 8.
  • Murine tumour cells which express the antigen for the antibody-drug conjugate are inoculated subcutaneously into the flank of immunocompetent mice, for example Balb/c or C57Bl/6 mice.1-10 million cells are detached from the cell culture, centrifuged and resuspended in medium ormedium/matrigel. The cell suspension is injected under the skin of the mouse. Within a few days, a tumour grows. Treatment is commenced after the tumour is established, at a tumour size of approximately 40 mm2. To examine the effect on laiger tumours, treatment may be initiated only at a tumour size of 50-100 mm2.
  • Treatment with A2DCs is carried out intraperitoneally or via the intravenous (i.v.) route into the tail vein of the mouse.
  • the A2DC is administered in a volume of 5 ml/kg.
  • the treatment protocol depends on the pharmacokinetics of the antibody. With the conjugates according to the invention, treatment is effected once or twice per week for 2 or 3 weeks as the standard. For highly efficacious compounds, a protocol with a single treatment may be employed. However, the treatment may also be continued, or a second cycle of three treatment days may follow at a later time. As standard, 8 animals are used per treatment group. In addition to the groups to which the active substances are administered, one group is treated as control group only with the buffer, according to the same protocol.
  • tumour area is measured regularly in two dimensions (length/width) using a caliper.
  • the tumour area is determined as length x width.
  • the ratio of the mean tumour area of the treatment group to that of the control group is stated as T/C area.
  • the syngeneic (mouse) tumor models CT26 and MC38 were investigated, both expressing the TWEAK-receptor as confirmed by immunohisto-chemistry.
  • the A2DC example II-11k-2658* was intraperitoneally injected in CT26 tumor bearing mice (Balb/c, female) as a single treatment at two different doses (2.5 and 5 mg/kg) and tumor growth was observed for approximately 3 weeks. As result a strong and dose-dependent tumor growth inhibition by 11k- 2658 could be demonstrated.

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Abstract

La présente invention concerne des conjugués anticorps-2 médicaments (A2DC) qui comprennent une pluralité de charges utiles d'inhibiteur de protéine Spindle de kinésine, ayant éventuellement différents profils de perméabilité, qui peuvent présenter des propriétés de libération de charge utile avantageuses dans un micro-environnement tumoral, des compositions pharmaceutiques, des procédés de préparation de ceux-ci, et leur utilisation pour le traitement, la prévention ou la gestion de maladies et d'états comprenant des troubles hyperprolifératifs, tels que le cancer chez l'homme et d'autres mammifères.
PCT/EP2023/082136 2022-11-17 2023-11-16 Conjugués anticorps-2 médicaments (a2dc) à groupes clivables par voie enzymatique WO2024105205A1 (fr)

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