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WO2014057118A1 - Conjugués anticorps anti-cd22 - pyrrolobenzodiazépine - Google Patents

Conjugués anticorps anti-cd22 - pyrrolobenzodiazépine Download PDF

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Publication number
WO2014057118A1
WO2014057118A1 PCT/EP2013/071347 EP2013071347W WO2014057118A1 WO 2014057118 A1 WO2014057118 A1 WO 2014057118A1 EP 2013071347 W EP2013071347 W EP 2013071347W WO 2014057118 A1 WO2014057118 A1 WO 2014057118A1
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Prior art keywords
conjugate according
group
antibody
alkyl
conjugate
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PCT/EP2013/071347
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English (en)
Inventor
Patricius Hendrikus Cornelis VAN BERKEL
Philip Wilson Howard
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Adc Therapeutics Sarl
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Publication of WO2014057118A1 publication Critical patent/WO2014057118A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • 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
    • A61K47/6867—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 a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to pyrrolobenzodiazepines (PBDs) having a labile C2 or N10 protecting group in the form of a linker to an antibody.
  • PBDs pyrrolobenzodiazepines
  • PBDs pyrrolobenzodiazepines
  • Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit, 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751 -758 (1992)), mazethramycin (Kuminoto, et al., J.
  • PBDs are of the general structure:
  • a particularly advantageous pyrrolobenzodiazepine compound is described by Gregson et al. (Chem. Commun. 1999, 797-798) as compound 1 , and by Gregson et al. (J. Med. Chem. 2001 , 44, 1 161 -1 174) as compound 4a.
  • This compound also known as SG2000, is shown below:
  • WO 2007/085930 describes the preparation of dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody.
  • the linker is present in the bridge linking the monomer PBD units of the dimer.
  • the present inventors have described dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, in WO 201 1/130613 and WO 201 1/130616.
  • the linker in these compounds is attached to the PBD core via the C2 position, and are generally cleaved by action of an enzyme on the linker group.
  • the linker in these compounds is attached to one of the available N10 positions on the PBD core, and are generally cleaved by action of an enzyme on the linker group.
  • ADC antibody-drug conjugates
  • cytotoxic or cytostatic agents i.e. drugs to kill or inhibit tumor cells in the treatment of cancer
  • cytotoxic or cytostatic agents i.e. drugs to kill or inhibit tumor cells in the treatment of cancer
  • systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells
  • Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • the present inventors have developed particular PBD dimer antibody conjugates.
  • a first aspect of the present invention comprises a conjugate of formula L - (D L ) P , where D L is of formula I or II:
  • L is an antibody (Ab) as defined below;
  • R 12 is selected from the group consisting of:
  • each of R 21 , R 22 and R 23 are independently selected from H, Ci -3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 12 group is no more than 5;
  • R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy;
  • R is selected from: H; Ci -3 saturated alkyl; C 2-3 alkenyl; C 2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
  • R and R are independently selected from H, F, C1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from Ci -4 alkyl amido and Ci -4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C1-4 alkyl ester;
  • R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , N HR, NRR', nitro, Me 3 Sn and halo;
  • R and R' are independently selected from optionally substituted Ci_i 2 alkyl, C 3 - 20 heterocyclyl and C 5-2 o aryl groups;
  • R 7 is selected from H, R, OH, OR, SH, SR, N H 2 , NHR, NHRR', nitro, Me 3 Sn and halo;
  • R" is a C 3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR N2 (where R N2 is H or C 1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
  • Y and Y' are selected from O, S, or NH;
  • R 6 , R 7 , R 9 are selected from the same groups as R 6 , R 7 and R 9 respectively;
  • R L1 is a linker for connection to the antibody (Ab);
  • R 11a is selected from OH, OR A , where R A is Ci -4 alkyl, and SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
  • R 20 and R 21 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
  • R 20 is selected from H and R c , where R c is a capping group
  • R 21 is selected from OH, OR A and SO z M;
  • R 2 is selected from the group consisting of:
  • R 15a and R 15b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
  • R 14 is selected from: H; C 1-3 saturated alkyl; C 2-3 alkenyl; C 2- 3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
  • R 2 is , where R 16a and R 16b are independently selected from H, F, Ci -4
  • R 22 is of formula Ilia, formula Illb or formula 111 c:
  • A is a C5-7 aryl group
  • Q 1 is a single bond
  • Q 2 is selected from a single bond and -Z-(CH 2 ) n -, where Z is selected from a single bond, O, S and NH and n is from 1 to 3;
  • R C1 , R C2 and R C3 are independently selected from H and unsubstituted Ci -2 alkyl;
  • Q is selected from 0-R L2' , S-R L2' and NR N -R L2 , and R N is selected from H, methyl and ethyl
  • R L2 is a linker for connection to the antibody (Ab);
  • R 10 and R 11 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
  • R 10 is H and R 11 is selected from OH, OR A and SO z M;
  • R 30 and R 31 either together form a double bond between the nitrogen and carbon atoms to which they are bound or;
  • R 30 is H and R 31 is selected from OH, OR A and SO z M.
  • the conjugate is selected from a conjugate of formula ConjA, ConjB, ConjC, ConjD and ConjE.
  • the subscript p in the formula I is an integer of from 1 to 20.
  • the Conjugates comprise an antibody (Ab) as defined below covalently linked to at least one Drug unit by a Linker unit.
  • the Ligand unit described more fully below, is a targeting agent that binds to a target moiety.
  • the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease.
  • the drug loading is represented by p, the number of drug molecules per antibody. Drug loading may range from 1 to 20 Drug units (D L ) per antibody.
  • D L Drug units
  • p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
  • a second aspect of the invention provides a method of making a conjugate according to the first aspect of the invention comprising conjugating a compound of formula l L or ll L :
  • R L1 is a linker suitable for conjugation to the antibody (Ab);
  • 2 2L is of formula llla L , formula lllb L or formula 111 c L :
  • Q L is selected from 0-R L2 , S-R L2 and NR N -R L2 , and R N is selected from H, methyl and ethyl L2
  • R N is selected from the group comprising H and C 1-4 alkyl
  • R L2 is a linker suitable for conjugation to the antibody (Ab);
  • the invention provides a method of making a conjugate selected from the group consisting of ConjA, ConjB, ConjC, ConjD and ConjE comprising conjugating a compound which is selected respectively from A:
  • W 201 1/130615 discloses compound 26: which is the parent compound of A.
  • Compound A comprises this PBD with a linker for attachment to a cell binding agent.
  • the cell binding agent provides a number of ethylene glycol moieties to provide solubility which is useful in the synthesis of conjugates.
  • WO 2010/043380 and WO 201 1/130613 disclose compound 30:
  • W 201 1/130613 also discloses compound 51 :
  • Compound B differs from compound 30 by only having a (CH 2 )3 tether between the PBD moieties, instead of a (CH 2 )5 tether, which reduces the lipophilicity of the released PBD dimer.
  • the linking group is attached to the C2-phenyl group in the para rather than meta
  • Compound C differs from this in two respects.
  • the cell binding agent provides an increased number of ethylene glycol moieties to provide solubility which is useful in the synthesis of conjugates, and the phenyl substiuent provide two rather than one oxygen atom, which also aids solubility.
  • Compound C's strucutre may also mean it binds more strongly in the minor groove.
  • Compounds A, B and C have two sp 2 centres in each C-ring, which may allow for stronger binding in the minor groove of DNA, than for compounds with only one sp 2 centre in each C- ring.
  • WO 201 1/130598 discloses compound 80:
  • Compound D differs from this by comprising an iodoacetamide group for linking to the cell binding agent.
  • This group may offer advantages over compound 80 with regards to its stability when bound to a cell binding agent (see below).
  • the malemide group in compound 80 can undergo a retro-Michael reaction, becoming unconjugated from the cell binding agent, and thus vunerable to scavenging by other thiol containing biological molecules, such as albumin and glutathione. Such unconjugation cannot occur with compound A.
  • the iodoacetamide group may avoid other unwanted side reactions.
  • Compound E differs from previously disclosed PBD dimers with a drug linker having a C2-3 endo-double bond, by having a smaller, less lipophilic C2 substituent, e.g. 4F-phenyl, propylene.
  • the conjugates of compound B are less likely to aggregate once synthesised. Such aggregation of conjugates can be measured by Size exclusion chromatography (SEC).
  • Both compound D and E have two sp 2 centres in each C-ring, which may allow for stronger binding in the minor groove of DNA, than for compounds with only one sp 2 centre in each C- ring.
  • the drug linkers disclosed in WO 2010/043880, WO 201 1/130613, WO 201 1/130598 and WO 201 1/130616 may be used in the present invention, and are incorporated herein by reference.
  • the drug linkers described herein may be synthesised as described in these disclosures. Detailed description of the invention
  • the present invention is suitable for use in providing a PBD compound to a preferred site in a subject.
  • the conjugate allows the release of an active PBD compound that does not retain any part of the linker. There is no stub present that could affect the reactivity of the PBD mpound. Thus ConjA would release the compound RelA:
  • the speficied link between the PBD dimer and the antibody, in the present invention is preferably stable extracellularly.
  • the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains linked to the drug moiety.
  • the linkers are stable outside the target cell and may be cleaved at some efficacious rate inside the cell.
  • An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e.
  • Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS.
  • Delivery of the compounds of formulae RelA, RelB, ReIC, RelD or RelE is achieved at the desired activation site of the conjugates of formulae ConjA, ConjB, ConjC, ConjD or ConjE by the action of an enzyme, such as cathepsin, on the linking group, and in particular on the valine-alanine dipeptide moiety.
  • an enzyme such as cathepsin
  • the antibody is an isolated humanized, anti-CD22 antibodies, having at least one antigen binding region derived from the high affinity VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 anti-CD22 antibodies, as well as anti-CD22 antibody compositions, conjugated version of these anti-CD22 antibodies, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants related thereto, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art.
  • the antibodies specifically neutralize human CD22 with high affinity.
  • the antibody is any one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015, or any protein or peptide molecule that comprises at least one complementarity determining region (CDR) (e.g., CDR1 , CDR2 or CDR3) of a heavy or light chain or a ligand binding portion thereof, derived from one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015, in combination with a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into the antibody.
  • CDR
  • the antibody is an anti-CD22 antibody comprising a light chain and a heavy chain, each of the chains comprising at least part of a human constant region and at least part of a variable region (v) derived from one or more of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 each of which has specificity to human CD22, said antibody binding with high affinity to an inhibiting and/or neutralizing epitope of human CD22.
  • v variable region
  • antibody also includes fragments or a derivative, such as one or more portions of the antibody chain, such as the heavy chain constant, joining, diversity or variable regions, or the light chain constant, joining or variable regions.
  • the antibody can comprise at least one specified portion of at least one complementarily determining region (CDR) (e.g., CDR1 , CDR2 or CDR3 of the heavy or light chain variable region) derived from an anti-CD22 antibody (as such term is defined herein), and/or at least one constant or variable framework region or any portion thereof.
  • CDR complementarily determining region
  • the antibody amino acid sequence can further optionally comprise at least one specified substitution, insertion or deletion as described herein or as known in the art.
  • Preferred antibodies include VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, and VM1015, as well as fragments and regions thereof.
  • the antibody is an isolated antibody or antibody fragment that binds to human CD22, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, or 64; a light chain variable region having the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32; and a constant region derived from one or more human antibodies.
  • the disclosure provides an isolated antibody or antibody fragment that binds to human CD22, comprising a heavy chain and light chain complementarity determining regions (CDRs) derived from the variable regions from one or more of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, and VM1015, and a constant region derived from one or more human antibodies.
  • the disclosure provides an antibody or fragment according to claim 1 or 2, wherein said antibody or fragment competitively inhibits in vivo the binding to human CD22 of an anti CD22 murine antibody.
  • Preferred antibodies are those that bind human CD22 and induce CD22 tyrosine
  • the antibody binds at least one specified epitope specific to human CD22 protein, subunit, fragment, portion or any combination thereof, for example as described by (Stein R, Belisle E, Hansen HJ,
  • the monoclonal antibody LL2 binds to the third immunoglobulin (Ig) repeat sequence in the extracellular domain of CD22 and is important for its modulatory effects on CD22 expressing B cell populations and lymphoma and leukemia cells.
  • the epitope can comprise at least one antibody binding region, which epitope is preferably comprised of at least 1 -5 amino acids of at least one portion thereof, such as but not limited to, at least one functional, extracellular, soluble, hydrophillic, external or cytoplasmic domain of human CD22 protein, or any portion thereof.
  • the antibody is an isolated mammalian anti-CD22 antibody, comprising at least one variable region from one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, and VM1015 and the nucleic acid sequences encoding them.
  • the antibody is an isolated mammalian anti-CD22 antibody, comprising either (i) all of the heavy chain complementarity determining regions (CDR) amino acid sequences derived from VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 and the nucleic acid sequences encoding them; or (ii) all of the light chain CDR amino acids sequences from one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 and the nucleic acid sequences encoding them.
  • CDR heavy chain complementarity determining regions
  • the antibody is an isolated mammalian anti-CD22 antibody, comprising at least one heavy chain or light chain CDR having the amino acid sequence derived from VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, and VM1015 and the nucleic acid sequences encoding them.
  • the antibody is an isolated mammalian chimeric, humanized or CDR-grafted anti-CD22 antibody, comprising at least one human CDR, or no human CDRs, wherein the antibody specifically binds at least one epitope comprising at least 1 -3 amino acids of an epitope of human CD22.
  • the antibody can optionally further bind CD22 with an affinity (K D of at least 10 "9 M, preferably at least 10 "10 M, and/or substantially neutralize at least one activity of at least one CD22 protein.
  • the antibody binds CD22 with an affinity (K Ds ) of at least 5 x 10 "10 M, preferably 5 x 10 "11 , more preferably 5 x 10 "12 and neutralizes human CD22.
  • an "Anti-CD22 antibody,” “Anti-CD22 antibody,” “Anti-CD22 antibody portion,” or “Anti-CD22 antibody fragment” and/or “Anti-CD22 antibody variant” and the like include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, containing at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof derived from at least one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 , in combination with a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, of non-murine origin, preferably of human origin, which can be incorporated into the antibody.
  • CDR complementarity determining region
  • Anti-CD22 antibody shall refer collectively or individually to the humanized monoclonal antibodies VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, and VM1015.
  • Such antibody is capable of modulating, decreasing, antagonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with at least one CD22 activity or binding, or with CD22 receptor activity or binding, in vitro, in situ and/or in vivo.
  • a suitable Anti-CD22 antibody, specified portion or variant can bind with high affinity to an inhibiting and/or neutralizing epitope of human CD22 recognized by at least one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 monoclonal antibody.
  • a suitable Anti- CD22 antibody, specified portion, or variant can also optionally affect at least one of CD22 activity or function, such as but not limited to, RNA, DNA or protein synthesis, CD22 release, CD22 receptor signaling, membrane CD22 cleavage, CD22 activity, CD22 production and/or synthesis.
  • CD22 activity or function such as but not limited to, RNA, DNA or protein synthesis, CD22 release, CD22 receptor signaling, membrane CD22 cleavage, CD22 activity, CD22 production and/or synthesis.
  • antibody is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof, each containing at least one CDR derived from an Anti-CD22.
  • Functional fragments include antigen-binding fragments that bind to a mammalian CD22.
  • antibody fragments capable of binding to CD22 or portions thereof including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab') 2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by this disclosure (see, e.g., Colligan, Immunology, supra).
  • Fab e.g., by papain digestion
  • Fab' e.g., by pepsin digestion and partial reduction
  • F(ab') 2 e.g., by pepsin digestion
  • facb e.g., by plasmin digestion
  • Antibody fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH-i domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • chimeric antibodies or “humanized” antibodies or “CDR-grafted” include any combination of the herein described Anti-CD22 Abs, or any CDR derived therefrom combined with one or more proteins or peptides derived from a non-murine, preferably, human antibody.
  • Chimeric or humanized antibodies include those wherein the CDR's are derived from one or more of the Anti-CD22 Abs described herein and at least a portion, or the remainder of the antibody is derived from one or more human antibodies.
  • the human part of the antibody may include the framework, C L , C H domains (e.g., Cm, C H 2, Cm), hinge, (V L , V H )) regions which are substantially non-immunogenic in humans.
  • the regions of the antibody that are derived from human antibodies need not have 100% identity with human antibodies.
  • the human amino acid residues as many of the human amino acid residues as possible are retained in order for the immunogenicity to be negligible, but the human residues may be modified as necessary to support the antigen binding site formed by the CDR's while simultaneously maximizing the humanization of the antibody.
  • Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans or other species relative to non-modified antibodies.
  • a humanized antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • Antibody humanization can be performed by, for example, synthesizing a combinatorial library comprising the six CDRs of a non-human target monoclonal antibody fused in frame to a pool of individual human frameworks.
  • a human framework library that contains genes representative of all known heavy and light chain human germline genes can be utilized.
  • the resulting combinatorial libraries can then be screened for binding to antigens of interest. This approach can allow for the selection of the most favorable combinations of fully human frameworks in terms of maintaining the binding activity to the parental antibody.
  • Humanized antibodies can then be further optimized by a variety of techniques. For full-length antibody molecules, the immunoglobulin genes can be obtained from genomic DNA or mRNA of hybridoma cell lines.
  • Antibody heavy and light chains are cloned in a mammalian vector system. Assembly is documented with double strand sequence analysis.
  • the antibody construct can be expressed in other human or mammalian host cell lines. The construct can then be validated by transient transfection assays and Western blot analysis of the expressed antibody of interest. Stable cell lines with the highest productivity can be isolated and screened using rapid assay methods.
  • the Anti-CD22 antibodies comprise any one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 antibodies or an antibody in which the variable region or CDRs are derived from any one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 antibody and the framework and constant regions of the antibody are derived from one or more human antibodies.
  • variable region or CDRs derived from the antibody preferably have from about 90% to about 100% identity with the variable region or CDRs of any one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015 antibody, although any and all modifications, including substitutions, insertions and deletions, are contemplated so long as the chimeric antibody maintains the ability to bind to and inhibit CD22.
  • the regions of the chimeric, humanized or CDR-grafted antibodies that are derived from human antibodies need not have 100% identity with the human antibodies.
  • Each of the heavy and light chain variable regions contain three CDRs that combine to form the antigen binding site.
  • the three CDRs are surrounded by four framework regions that primarily function to support the CDRs.
  • the sequences of the CDRs within the sequences of the variable regions of the heavy and light chains can be identified by computer-assisted alignment according to Kabat et al. (1987) in Sequences of Proteins of Immunological Interest, 4 th ed., United States Department of Health and Human Services, U.S. Government Printing Office, Washington, D.C., or by molecular modeling of the variable regions, for example utilizing the ENCAD program as described by Levitt (1983) J. Mol. Biol. 168:595.
  • the CDRs are derived from any one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010,
  • VM101 1 VM1012, VM1013, VM1014, or VM1015.
  • Determination of the heavy chain CDRs and light chain CDRs is well within the skill of one in the art. See, for example,
  • the sequences of the CDRs of the Anti-CD22 antibody may be modified by insertions, substitutions and deletions to the extent that the CDR-grafted antibody maintains the ability to bind to and inhibit human CD22.
  • the ordinarily skilled artisan can ascertain the maintenance of this activity by performing the functional assays described herein below.
  • VM1000 comprises light chain VM1000LC (SEQ ID NO: 1 ) and heavy chain
  • Clone VM1001 comprises light chain VM1001 LC (SEQ ID NO: 2) and heavy chain VM1001 HC (SEQ ID NO: 34).
  • Clone VM1002 comprises light chain VM1002LC (SEQ ID NO: 3) and heavy chain VM1002HC (SEQ ID NO: 35).
  • Clone VM1003 comprises light chain VM1003LC (SEQ ID NO: 4) and heavy chain VM1003HC (SEQ ID NO: 36).
  • Clone VM1004 comprises light chain VM1004LC (SEQ ID NO: 5) and heavy chain VM1004HC (SEQ ID NO: 37).
  • Clone VM1005 comprises light chain VM1005LC (SEQ ID NO: 6) and heavy chain VM1005HC (SEQ ID NO: 38).
  • Clone VM1006 comprises light chain VM1006LC (SEQ ID NO: 7) and heavy chain VM1006HC (SEQ ID NO: 39).
  • Clone VM1007 comprises light chain VM1007LC (SEQ ID NO: 8) and heavy chain VM1007HC (SEQ ID NO: 40).
  • Clone VM1008 comprises light chain VM1008LC (SEQ ID NO: 9) and heavy chain VM1008HC (SEQ ID NO: 41 ).
  • Clone VM1009 comprises light chain VM1009LC (SEQ ID NO: 10) and heavy chain VM1009HC (SEQ ID NO: 42).
  • Clone VM1010 comprises light chain VM1010LC (SEQ ID NO: 1 1 ) and heavy chain VM1010HC (SEQ ID NO: 43).
  • Clone VM101 1 comprises light chain VM101 1 LC (SEQ ID NO: 12) and heavy chain VM101 1 HC (SEQ ID NO: 44).
  • Clone VM1012 comprises light chain VM1012LC (SEQ ID NO: 13) and heavy chain VM1012HC (SEQ ID NO: 45).
  • Clone VM1013 comprises light chain VM1013LC (SEQ ID NO: 14) and heavy chain VM1013HC (SEQ ID NO: 46).
  • Clone VM1014 comprises light chain VM1014LC (SEQ ID NO: 15) and heavy chain VM1014HC (SEQ ID NO: 47).
  • Clone VM1015 comprises light chain VM1015LC (SEQ ID NO: 16) and heavy chain
  • Human genes which encode the constant (C) regions of the humanized antibodies, fragments and regions can be derived from a human fetal liver library, by known methods. Human C region genes can be derived from any human cell including those which express and produce human immunoglobulins.
  • the human C H region can be derived from any of the known classes or isotypes of human H chains, including ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and subtypes thereof, such as G1 , G2, G3 and G4. Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of C H region will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • the C H region is derived from gamma 1 (lgG1 ).
  • the human C L region can be derived from either human L chain isotype, kappa or lambda, preferably kappa.
  • Genes encoding human immunoglobulin C regions are obtained from human cells by standard cloning techniques (Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., eds. Current Protocols in Molecular Biology (1987-1993)).
  • Human C region genes are readily available from known clones containing genes representing the two classes of L chains, the five classes of H chains and subclasses thereof.
  • Chimeric antibody fragments such as F(ab 1 ) 2 and Fab, can be prepared by designing a chimeric H chain gene which is appropriately truncated.
  • a chimeric gene encoding an H chain portion of an F(ab 1 ) 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • humanized antibodies, fragments and regions are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of the Anti CD22 specific antibody, and joining these DNA segments to DNA segments including C H and C L regions, respectively, to produce full length immunoglobulin-encoding genes.
  • variable regions of the antibody may be modified by insertions, substitutions and deletions to the extent that the chimeric antibody maintains the ability to bind to and inhibit human CD22.
  • the ordinarily skilled artisan can ascertain the
  • variable regions can have, for example, from about 50% to about 100% homology to the variable regions of SEQ ID NOS: 1 -64.
  • variable regions of the antibody have from about 80% to about 100% homology to the variable regions of SEQ ID NOS: 1 -64.
  • variable regions have from about 90% to about 100% homology to the variable regions of SEQ ID NOS: 1-64.
  • preferred anti-CD22 Mabs of the disclosure comprise variable light chain regions having 95%, 96%, 97%, 98% or 99% amino acid sequence homology to SEQ ID NO: 17-32 and further comprise variable heavy chain regions having 95%, 96%, 97%, 98% or 99% amino acid sequence homology to SEQ ID NO: 49-64.
  • preferred anti-CD22 Mabs of the disclosure comprise a variable light chain region selected from one of SEQ ID NO: 1-16. In another specific aspect, preferred anti-CD22 Mabs of the disclosure comprise a variable heavy chain region selected from one of SEQ ID NO: 33-48.
  • Methods for engineering or humanizing non-human or human antibodies can be used and are well known in the art.
  • a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import” residues, which are typically taken from an "import” variable, constant or other domain of a known human sequence.
  • Known human Ig sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;
  • Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
  • Generally part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.
  • Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties.
  • humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Humanization or engineering the antibody can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151 : 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151 :2623 (1993), U.S. Pat.
  • the human constant region of the humanized antibody can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
  • IgG IgG, IgA, IgM, IgE, IgD, etc.
  • the human constant region comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, lgG1 , lgG2, lgG3 or lgG4.
  • the anti-human CD22 human antibody comprises an lgG1 heavy chain and a lgG1 K light chain.
  • the isolated anti-CD22 antibodies described herein comprise antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide as well as.
  • the antibody or antigen-binding fragment binds human CD22 and, thereby partially or substantially neutralizes at least one biological activity of the protein.
  • the antibody, or specified portion or variant thereof partially or preferably substantially neutralizes at least one biological activity of at least one CD22 protein or fragment and thereby inhibit activities mediated through the binding of CD22 to the CD22 receptor or through other CD22- dependent or mediated mechanisms.
  • neutralizing antibody refers to an antibody that can inhibit an CD22-dependent activity by about 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay.
  • the capacity of an Anti-CD22 antibody to inhibit a CD22-dependent activity is preferably assessed by at least one suitable CD22 protein or receptor assay, as described herein and/or as known in the art.
  • At least one antibody described herein binds at least one specified epitope specific to at least one CD22 protein, subunit, fragment, portion or any combination thereof.
  • the at least one epitope can comprise at least one antibody binding region that comprises at least one portion of the protein, which epitope is preferably comprised of at least one extracellular, soluble, hydrophillic, external or cytoplasmic portion of the protein.
  • the human antibody or antigen-binding fragment will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDR1 , CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR4, CDR5 and CDR6) or variant of at least one light chain variable region, derived from an anti-CD22 Ab described herein.
  • the antibody or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e., CDR1 , CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1 , 2 and/or 3.
  • the antibody or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDR4, CDR5 and/or CDR6) having the amino acid sequence of the corresponding CDRs 4, 5 and/or 6.
  • CDR4 light chain CDR
  • CDR5 light chain CDR5
  • CDR6 having the amino acid sequence of the corresponding CDRs 4, 5 and/or 6.
  • the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen-binding fragment have the amino acid sequence of the corresponding CDR of at least one of VM1000, VM1001 , VM1002, VM1003, VM1004, VM1005, VM1006, VM1007, VM1008, VM1009, VM1010, VM101 1 , VM1012, VM1013, VM1014, or VM1015.
  • Such antibodies can be prepared by chemically joining together the various portions (e.g., CDRs, framework) of the antibody using conventional techniques, by preparing and expressing a (i.e., one or more) nucleic acid molecule that encodes the antibody using conventional techniques of recombinant DNA technology or by using any other suitable method and using any of the possible redundant codons that will result in expression of an anti-CD22 antibody.
  • a (i.e., one or more) nucleic acid molecule that encodes the antibody using conventional techniques of recombinant DNA technology or by using any other suitable method and using any of the possible redundant codons that will result in expression of an anti-CD22 antibody.
  • Liquid phase synthesis of combinatorial variable domain humanized libraries for the light chain and the heavy chain can be employed.
  • the assembly of a humanized light chain (LC) variable domain library for example, contains human light chain frameworks (FW) and non- human complementarity determining regions (CDR).
  • the library is assembled by, for example, by using stepwise liquid phase ligation of FW and CDR DNA fragments.
  • the libraries are assembled by using stepwise liquid phase ligation of FW and CDR DNA fragments in the order of FW1 -CDR1 -FW2-CDR2-FW3-CDR3 by techniques known to one of skill in the art. For example, by the techniques of one or more of the following references, each of which is incorporated herein by reference. Lo, B.K., 2003, Antibody humanization by CDR grafting. Antibody Engineering, Methods and protocols. Edit by Benny K. C.
  • Antibodies that bind to human CD22 and that comprise the defined heavy or light chain variable region or CDR regions can be prepared using suitable methods, such as phage display (Katsube, Y., et al., Int J. Mol. Med, 1 (5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein.
  • the antibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
  • the "antibody” may be a full-length antibody, an antigen-binding fragment, an immunoglobulin chain and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
  • Anti-CD22 antibodies can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can bind human CD22 with high affinity (e.g., K D less than or equal to about 10 "9 M).
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given Anti-CD22 antibody, fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , such as 1 -30 or any range or value therein, as specified herein.
  • Amino acids in an Anti-CD22 antibody that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning
  • Anti-CD22 antibodies can include, but are not limited to, at least one portion, sequence or combination selected from 5 to all of the contiguous amino acids of at least one of CDRs derived from SEQ ID NOS: 17-32 and 49-64.
  • An Anti-CD22 antibody can further optionally comprise a polypeptide of at least one of 70- 100% of the contiguous amino acids of the CDRs derived from at least one of SEQ ID NOS: 17-32 and 49-64.
  • the anti-CD22 antibody comprises a polypeptide of 95-99% sequence homology to SEQ ID NO: 1 -16.
  • the anti-CD22 antibody comprises a polypeptide of 95-99% sequence homology to SEQ ID NO: 33-48.
  • the amino acid sequence of an immunoglobulin chain, or portion thereof has about 70-100% identity (e.g., 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the amino acid sequence of at least one of SEQ ID NOS: 17- 32 or 49-64.
  • 70-100% amino acid identity i.e., 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein
  • the anti-CD22 antibody comprises a polypeptide of 95-99% sequence homology to SEQ ID NO: 17-32 or 49-64.
  • Exemplary heavy chain and light chain variable regions sequences are provided in SEQ ID NOS: 1-64.
  • the antibodies described herein, or specified variants thereof can comprise any number of contiguous amino acid residues from an antibody described herein, wherein that number is selected from the group of integers consisting of from 10-100% of the number of contiguous residues in an Anti-CD22 antibody.
  • this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein.
  • the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as at least 2, 3, 4, or 5.
  • the antibody includes at least one biologically active antibody.
  • Biologically active antibodies have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%- 100% of that of the native (non-synthetic), endogenous or related and known antibody.
  • the antibody may be a human antibody or antigen-binding fragment, as described herein, which is modified by the covalent attachment of an organic moiety. Such modification can produce an antibody or antigen-binding fragment with improved
  • the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
  • the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • the modified antibodies and antigen-binding fragments can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody.
  • Each organic moiety that is bonded to an antibody or antigen-binding fragment described herein can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
  • fatty acid encompasses mono-carboxylic acids and di-carboxylic acids.
  • Hydrophilic polymers suitable for modifying antibodies described herein can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
  • polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
  • carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
  • polymers of hydrophilic amino acids e.g., polylysine,
  • the hydrophilic polymer that modifies the antibody described herein has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
  • PEG5000 and PEG 2 o,ooo wherein the subscript is the average molecular weight of the polymer in Daltons, can be used.
  • the hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups.
  • Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods.
  • a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with ⁇ , ⁇ -carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
  • an activated carboxylate e.g., activated with ⁇ , ⁇ -carbonyl diimidazole
  • Fatty acids and fatty acid esters suitable for modifying antibodies described herein can be saturated or can contain one or more units of unsaturation.
  • Fatty acids that are suitable for modifying antibodies described herein include, for example, n-dodecanoate (C 12 , laurate), n- tetradecanoate (C 14 , myristate), n-octadecanoate (C 18 , stearate), n-eicosanoate (C 2 o, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C 3 o), n-tetracontanoate (C 40 ), cis- ⁇ 9-octadecanoate (Ci 8 , oleate), all cis- ⁇ 5,8, 1 1 , 14-eicosatetraenoate (C 2 o,
  • Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group.
  • the lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms.
  • modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents.
  • An “activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
  • amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N- hydroxysuccinimidyl esters (NHS), and the like.
  • Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2- nitrobenzoic acid thiol (TNB-thiol), and the like.
  • An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
  • Suitable methods to introduce activating groups into molecules are known in the art (see for example,
  • An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
  • organic group e.g., hydrophilic polymer, fatty acid, fatty acid ester
  • linker moiety for example a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
  • Suitable linker moieties include, for example, tetraethylene glycol,— (CH 2 ) 3 ⁇ , ⁇ NH ⁇ (CH 2 )6-NH ⁇ , ⁇ (CH 2 ) 2 ⁇ NH ⁇ and -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH- NH-.
  • Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc- diaminohexane) with a fatty acid in the presence of 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.
  • a mono-Boc-alkyldiamine e.g., mono-Boc-ethylenediamine, mono-Boc- diaminohexane
  • EDC 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid.
  • TFA trifluoroacetic acid
  • the modified antibodies can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent.
  • the organic moieties can be bonded to the antibody in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
  • Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or antigen-binding fragment.
  • the reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody described herein.
  • Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an antibody described herein can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:41 1-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1 ): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).
  • suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem
  • the antibodies described herein can bind human CD22 with a wide range of affinities (K D ).
  • at least one human mAb described herein can optionally bind human CD22 with high affinity.
  • a mAb can bind human CD22 with a K D equal to or less than about 10 "7 M, such as but not limited to, 0.1 -9.9 (or any range or value therein) X 10 "7 , 10 "8 , 10 "9 , 10 "10 , 10 "11 , 10 "12 , 10 “13 or any range or value therein.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method.
  • the measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH).
  • affinity and other antigen-binding parameters are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, such as the buffer described herein.
  • Anti-CD22 antibodies useful in the methods and compositions described herein are characterized by high affinity binding to CD22 and optionally and preferably having low toxicity.
  • an antibody, specified fragment or variant described herein, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity is useful.
  • the antibodies that can be used are optionally characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity.
  • Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties can contribute to the therapeutic results achieved. "Low
  • immunogenicity is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al., Lancet 344:1 125-1 127 (1994), entirely incorporated herein by reference).
  • Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for at least one CD22 protein, the other one is for any other antigen.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)).
  • the pharmaceutically acceptable cation may be inorganic or organic.
  • Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + .
  • Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca 2+ and Mg 2+ .
  • Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH 4 + ) and substituted ammonium ions (e.g. NH 3 R + , NH 2 R2 + , NHR 3 + , NR 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine,
  • phenylbenzylamine choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • amino acids such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • substituted refers to a parent group which bears one or more substituents.
  • substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group.
  • substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
  • Ci-12 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • C1-4 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • alkyl includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
  • saturated alkyl groups include, but are not limited to, methyl (Ci), ethyl (C 2 ), propyl (C 3 ), butyl (C 4 ), pentyl (C 5 ), hexyl (C 6 ) and heptyl (C 7 ).
  • saturated linear alkyl groups include, but are not limited to, methyl (Ci), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ) and n-heptyl (C 7 ).
  • saturated branched alkyl groups include iso-propyl (C 3 ), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C 5 ), and neo-pentyl (C 5 ).
  • C2-12 Alkenyl The term "C2-12 alkenyl" as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
  • C2-12 alkynyl refers to an alkyl group having one or more carbon-carbon triple bonds.
  • unsaturated alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH) and 2-propynyl (propargyl, -CH 2 -C ⁇ CH).
  • C3-12 cycloalkyl refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
  • cycloalkyl groups include, but are not limited to, those derived from:
  • C3-20 heterocyclyl refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms.
  • each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
  • the prefixes e.g. C3-20, C3-7, C 5- 6, etc.
  • the prefixes denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.
  • C 5-6 heterocyclyl as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
  • monocyclic heterocyclyl groups include, but are not limited to, those derived from:
  • N 2 imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline
  • NiOi tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ),
  • O1S1 oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and,
  • substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C 5 ), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C 6 ), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose,
  • C5-2 0 aryl The term "C5-2 0 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms.
  • C 5-7 aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms
  • C5-1 0 aryl pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms.
  • each ring has from 5 to 7 ring atoms.
  • the prefixes denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.
  • C 5-6 aryl as used herein, pertains to an aryl group having 5 or 6 ring atoms.
  • the ring atoms may be all carbon atoms, as in "carboaryl groups”.
  • carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C 6 ), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (Ci 4 ), naphthacene (Ci 8 ), and pyrene (Ci 6 ).
  • aryl groups which comprise fused rings, at least one of which is an aromatic ring include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1 H-indene) (Cg), indene (C 9 ), isoindene (C 9 ), tetraline (1 ,2,3,4-tetrahydronaphthalene (C10),
  • the ring atoms may include one or more heteroatoms, as in "heteroaryl groups".
  • heteroaryl groups include, but are not limited to, those derived from:
  • N3O1 oxatriazole (C 5 );
  • N1S1 thiazole (C 5 ), isothiazole (C 5 );
  • N 2 imidazole (1 ,3-diazole) (C 5 ), pyrazole (1 ,2-diazole) (C 5 ), pyridazine (1 ,2-diazine) (C 6 ), pyrimidine (1 ,3-diazine) (C 6 ) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C 6 );
  • N 3 triazole (C 5 ), triazine (C 6 ); and,
  • heteroaryl which comprise fused rings, include, but are not limited to:
  • C 9 (with 2 fused rings) derived from benzofuran (Oi), isobenzofuran (Oi), indole (N-i), isoindole (N ⁇ , indolizine (N ⁇ , indoline (N ⁇ , isoindoline (N ⁇ , purine (N 4 ) (e.g., adenine, guanine), benzimidazole (N 2 ), indazole (N 2 ), benzoxazole (NiOi), benzisoxazole (NiOi), benzodioxole (0 2 ), benzofurazan (N ⁇ ), benzotriazole (N 3 ), benzothiofuran (S ⁇ , benzothiazole ( ⁇ ), benzothiadiazole (N 2 S);
  • Ci 3 (with 3 fused rings) derived from carbazole (Ni), dibenzofuran (Oi),
  • Ci 4 (with 3 fused rings) derived from acridine (N-i), xanthene (Oi), thioxanthene (Si), oxanthrene (0 2 ), phenoxathiin (O 1 S 1 ), phenazine (N 2 ), phenoxazine (N- ⁇ - ⁇ ), phenothiazine (N-I S-I), thianthrene (S 2 ), phenanthridine (N-i), phenanthroline (N 2 ), phenazine (N 2 ).
  • Halo -F, -CI, -Br, and -I.
  • Ether -OR, wherein R is an ether substituent, for example, a C 1-7 alkyl group (also referred to as a C 1-7 alkoxy group, discussed below), a C 3-20 heterocyclyl group (also referred to as a C 3-20 heterocyclyloxy group), or a C 5 - 2 o aryl group (also referred to as a C 5-2 o aryloxy group), preferably a Ci -7 alkyl group.
  • Alkoxy -OR, wherein R is an alkyl group, for example, a Ci_ 7 alkyl group.
  • Ci -7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu)
  • Acetal -CH(OR 1 )(OR 2 ), wherein R 1 and R 2 are independently acetal substituents, for example, a Ci -7 alkyl group, a C3-2 0 heterocyclyl group, or a C 5 -2o aryl group, preferably a Ci -7 alkyl group, or, in the case of a "cyclic" acetal group, R 1 and R 2 , taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • acetal groups include, but are not limited to, -CH(OMe) 2 , -CH(OEt) 2 , and -CH(OMe)(OEt).
  • Hemiacetal -CH(OH)(OR 1 ), wherein R 1 is a hemiacetal substituent, for example, a C 1-7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1 -7 alkyl group.
  • hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and - CH(OH)(OEt).
  • Ketal -CR(OR 1 )(OR 2 ), where R 1 and R 2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a Ci -7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • ketal groups include, but are not limited to, -C(Me)(OMe) 2 , -C(Me)(OEt) 2 , -C(Me)(OMe)(OEt), -C(Et)(OMe) 2 , -C(Et)(OEt) 2 , and -C(Et)(OMe)(OEt).
  • R 1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a Ci -7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • hemiacetal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and
  • Imino (imine): NR, wherein R is an imino substituent, for example, hydrogen, Ci -7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably hydrogen or a Ci -7 alkyl group.
  • R is an acyl substituent, for example, a Ci_ 7 alkyl group (also referred to as Ci -7 alkylacyl or Ci -7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C 5 -2o aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group.
  • a Ci_ 7 alkyl group also referred to as Ci -7 alkylacyl or Ci -7 alkanoyl
  • C3-20 heterocyclyl group also referred to as C3-20 heterocyclylacyl
  • C 5 -2o aryl group also referred to as C5-20 arylacyl
  • Carboxy (carboxylic acid): -C( 0)OH.
  • acyloxy (reverse ester): -OC( 0)R, wherein R is an acyloxy substituent, for example, a C 1-7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1-7 alkyl group.
  • R is an acyloxy substituent, for example, a C 1-7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1-7 alkyl group.
  • Oxycarboyloxy: -OC( 0)OR, wherein R is an ester substituent, for example, a Ci -7 alkyl group, a C 3-2 o heterocyclyl group, or a C5-20 aryl group, preferably a Ci -7 alkyl group.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a Ci -7 alkyl group (also referred to as Ci -7 alkylamino or di-Ci -7 alkylamino), a C3-20 heterocyclyl group, or a C 5- 2o aryl group, preferably H or a Ci -7 alkyl group, or, in the case of a "cyclic" amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • a Ci -7 alkyl group also referred to as Ci -7 alkylamino or di-Ci -7 alkylamino
  • C3-20 heterocyclyl group or a C 5- 2o aryl group, preferably H or a Ci -7 alkyl group
  • R 1 and R 2 taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • Amino groups may be primary (-NH 2 ), secondary (-NHR 1 ), or tertiary (-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ).
  • Examples of amino groups include, but are not limited to, -NH 2 ,
  • cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
  • Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C( 0)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • Thioamido (thiocarbamyl): -C( S)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • Acylamido (acylamino): -NR 1 C( 0)R 2 , wherein R 1 is an amide substituent, for example, hydrogen, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably hydrogen or a C 1-7 alkyl group, and R 2 is an acyl substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 oaryl group, preferably hydrogen or a C 1-7 alkyl group.
  • R 1 and R 2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
  • R 2 and R 3 are independently amino substituents, as defined for amino groups, and R 1 is a ureido substituent, for example, hydrogen, a Ci_ 7 alkyl group, a C3-2 0 heterocyclyl group, or a C 5 -2o aryl group, preferably hydrogen or a Ci -7 alkyl group.
  • ureido groups include, but are not limited to, -NHCONH 2 , -NHCONHMe, -NHCONHEt, -NHCONMe 2 , -NHCONEt 2 , -NMeCONH 2 , -NMeCONHMe, -NMeCONHEt, - NMeCONMe 2 , and -NMeCONEt 2 .
  • Tetrazolyl a five membered aromatic ring having four nitrogen atoms and one carbon atom
  • Imino: NR, wherein R is an imino substituent, for example, for example, hydrogen, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group.
  • Isocyano -NC. Cyanato: -OCN. Isocyanato: -NCO.
  • Ci -7 alkylthio groups include, but are not limited to,
  • Disulfide -SS-R, wherein R is a disulfide substituent, for example, a Ci -7 alkyl group, a C3-2 0 heterocyclyl group, or a C 5- 2o aryl group, preferably a Ci -7 alkyl group (also referred to herein as Ci -7 alkyl disulfide).
  • R is a disulfide substituent, for example, a Ci -7 alkyl group, a C3-2 0 heterocyclyl group, or a C 5- 2o aryl group, preferably a Ci -7 alkyl group (also referred to herein as Ci -7 alkyl disulfide).
  • Ci -7 alkyl disulfide groups include, but are not limited to,
  • Sulfine (sulfinyl, sulfoxide): -S( 0)R, wherein R is a sulfine substituent, for example, a C 1-7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1-7 alkyl group.
  • R is a sulfine substituent, for example, a C 1-7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1-7 alkyl group.
  • Sulfonate (sulfonic acid ester): -S( 0) 2 OR, wherein R is a sulfonate substituent, for example, a Ci-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • R is a sulfonate substituent, for example, a Ci-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • R is a sulfinyloxy substituent, for example, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • R is a sulfonyloxy substituent, for example, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5 - 2 o aryl group, preferably a Ci -7 alkyl group.
  • R is a sulfate substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a C 1-7 alkyl group.
  • R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • R 1 is an amino substituent, as defined for amino groups.
  • R 1 is an amino substituent, as defined for amino groups
  • R is a sulfonamino substituent, for example, a C -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a C 1-7 alkyl group.
  • R 1 is an amino substituent, as defined for amino groups
  • R is a sulfinamino substituent, for example, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a Ci -7 alkyl group.
  • R is a phosphino substituent, for example, -H, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably -H, a Ci -7 alkyl group, or a C 5-2 o aryl group.
  • Examples of phosphino groups include, but are not limited to, -PH 2 , -P(CH 3 ) 2 , -P(CH 2 CH 3 ) 2 , -P(t-Bu) 2 , and -P(Ph) 2 .
  • R is a phosphinyl substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably a C 1-7 alkyl group or a C 5-20 aryl group.
  • R is a phosphonate substituent, for example, -H, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably -H, a C1-7 alkyl group, or a C 5 - 2 o aryl group.
  • Phosphate (phosphonooxy ester): -OP( 0)(OR) 2 , where R is a phosphate substituent, for example, -H, a Ci -7 alkyl group, a C 3-2 o heterocyclyl group, or a C 5-2 o aryl group, preferably -H, a C1-7 alkyl group, or a C 5 - 2 o aryl group.
  • Phosphorous acid -OP(OH) 2 .
  • Phosphite -OP(OR) 2
  • R is a phosphite substituent, for example, -H, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably -H, a C 1-7 alkyl group, or a C 5-20 aryl group.
  • phosphite groups include, but are not limited to, -OP(OCH 3 ) 2 ,
  • Phosphoramidite -OP(OR 1 )-NR 2 2 , where R 1 and R 2 are phosphoramidite substituents, for example, -H, a (optionally substituted) Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably -H, a Ci -7 alkyl group, or a C 5-2 o aryl group.
  • R 1 and R 2 are phosphoramidite substituents, for example, -H, a (optionally substituted) Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-2 o aryl group, preferably -H, a Ci -7 alkyl group, or a C 5-2 o aryl group.
  • phosphoramidite groups include, but are not limited to, -OP(OCH 2 CH 3 )-N(CH 3 ) 2 ,
  • C 3- i 2 alkylene refers to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated.
  • alkylene includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
  • Examples of linear saturated C 3- i 2 alkylene groups include, but are not limited to, -(CH 2 ) n - where n is an integer from 3 to 12, for example, -CH 2 CH 2 CH 2 - (propylene),
  • Examples of branched saturated C 3 -i 2 alkylene groups include, but are not limited to, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH2-, -CH 2 CH(CH 3 )CH 2 -,
  • C 3- i 2 cycloalkylenes examples include, but are not limited to, cyclopentylene (e.g. cyclopent-1 ,3-ylene), and cyclohexylene
  • C 3- i 2 cycloalkylenes examples include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1 ,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1 ,4-ylene; 3-cyclohexen-1 ,2-ylene; 2,5-cyclohexadien- 1 ,4-ylene).
  • Carbamate nitrogen protecting group the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure: wherein R' 10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T.W. and Wuts, G.M., Protective Groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
  • Hemi-aminal nitrogen protecting group the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure: wherein R' 10 is R as defined above.
  • the groups Carbamate nitrogen protecting group and Hemi-aminal nitrogen protecting group may be jointly termed a "nitrogen protecting group for synthesis”.
  • the present invention provides a conjugate comprising a PBD compound connected to the antibody via a Linker Unit.
  • the conjugate comprises the antibody connected to a spacer connecting group, the spacer connected to a trigger, the trigger connected to a self-immolative linker, and the self-immolative linker connected to the N10 position of the PBD compound.
  • a conjugate is illustrated below:
  • R L may be either R L1 or R L2 .
  • D is D L with R L or R L2' removed.
  • the present invention is suitable for use in providing a PBD compound to a preferred site in a subject.
  • the conjugate allows the release of an active PBD compound that does not retain any part of the linker. There is no stub present that could affect the reactivity of the PBD compound.
  • the linker attaches the antibody to the PBD drug moiety D through covalent bond(s).
  • the linker is a bifunctional or multifunctional moiety which can be used to link one or more drug moiety (D) and an antibody unit (Ab) to form antibody-drug conjugates (ADC).
  • the linker (R L ) may be stable outside a cell, i.e. extracellular, or it may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions.
  • Antibody-drug conjugates (ADC) can be conveniently prepared using a linker having reactive functionality for binding to the drug moiety and to the antibody.
  • N-terminus or amino acid side chain such as lysine, of the antibody (Ab) can form a bond with a functional group of a linker or spacer reagent, PBD drug moiety (D) or drug-linker reagent (D L , D -R L ), where R L can be
  • the linkers of the ADC preferably prevent aggregation of ADC molecules and keep the ADC freely soluble in aqueous media and in a monomeric state.
  • the linkers of the ADC are preferably stable extracellularly.
  • the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains linked to the drug moiety.
  • the linkers are stable outside the target cell and may be cleaved at some efficacious rate inside the cell.
  • An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered or transported to its targetted site; and (iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect of the PBD drug moiety.
  • Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the
  • bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G.T. (1996)
  • the linker may be substituted with groups which modulate aggregation, solubility or reactivity.
  • a sulfonate substituent may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or the drug moiety, or facilitate the coupling reaction of Ab-L with D L , or D L -L with Ab, depending on the synthetic route employed to prepare the ADC.
  • L-R L is a g
  • Ab is the antibody (L)
  • L 1 is a linker
  • A is a connecting group connecting L 1 to the antibody
  • L 1 or L 2 is a cleavable linker.
  • L 1 is preferably the cleavable linker, and may be referred to as a trigger for activation of the linker for cleavage.
  • L 1 and L 2 can vary widely. These groups are chosen on the basis of their cleavage characteristics, which may be dictated by the conditions at the site to which the conjugate is delivered. Those linkers that are cleaved by the action of enzymes are preferred, although linkers that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. Linkers that are cleavable under reducing or oxidising conditions may also find use in the present invention.
  • pH e.g. acid or base labile
  • temperature or upon irradiation e.g. photolabile
  • L 1 may comprise a contiguous sequence of amino acids.
  • the amino acid sequence may be the target substrate for enzymatic cleavage, thereby allowing release of L-R L from the N10 position.
  • L 1 is cleavable by the action of an enzyme.
  • the enzyme is an esterase or a peptidase.
  • the enzyme cleaves the bond between L 1 and L 2 .
  • L 1 and L 2 where present, may be connected by a bond selected from:
  • An amino group of L 1 that connects to L 2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
  • a carboxyl group of L 1 that connects to L 2 may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
  • a hydroxyl group of L 1 that connects to L 2 may be derived from a hydroxyl group of an amino acid side chain, for example a serine amino acid side chain.
  • amino acid side chain includes those groups found in: (i) naturally occurring amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids such as ornithine and citrulline; (iii) unnatural amino acids, beta-amino acids, synthetic analogs and derivatives of naturally occurring amino acids; and (iv) all enantiomers, diastereomers, isomerically enriched, isotopically labelled (e.g. 2 H, 3 H, 14 C, 15 N), protected forms, and racemic mixtures thereof.
  • naturally occurring amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine
  • the phenylene ring is optionally substituted with one, two or three substituents as described herein.
  • the phenylene group is optionally substituted with halo, N0 2 , R or OR.
  • Y is NH.
  • n is 0 or 1 .
  • n is 0.
  • the self-immolative linker may be referred to as a p-aminobenzylcarbonyl linker (PABC).
  • L is the activated form of the remaining portion of the linker.
  • the group L * is a linker L 1 as described herein, which may include a dipeptide group.
  • Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted. In one embodiment, the phenylene ring having the Y substituent is unsubstituted and the phenylene ring not having the Y substituent is optionally substituted.
  • E is O, S or NR
  • D is N, CH, or CR
  • F is N, CH, or CR.
  • D is N.
  • D is CH.
  • E is O or S.
  • F is CH.
  • the linker is a cathepsin labile linker.
  • L 1 comprises a dipeptide
  • the dipeptide may be represented as -NH-X X 2 -CO-, where -NH- and -CO- represent the N- and C-terminals of the amino acid groups Xi and X 2 respectively.
  • the amino acids in the dipeptide may be any combination of natural amino acids.
  • the linker is a cathepsin labile linker
  • the dipeptide may be the site of action for cathepsin-mediated cleavage.
  • CO and NH may represent that side chain
  • the group -X X 2 - in dipeptide is selected from:
  • the group -XrX 2 - in dipeptide, -NH-XrX 2 -CO-, is selected from:
  • the group -Xi-X 2 - in dipeptide, - ⁇ -X X ⁇ CO-, is -Phe-Lys- or -Val-Ala-.
  • dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
  • the amino acid side chain is derivatised, where appropriate.
  • an amino group or carboxy group of an amino acid side chain may be derivatised.
  • an amino group NH 2 of a side chain amino acid, such as lysine is a derivatised form selected from the group consisting of NHR and NRR'.
  • a carboxy group COOH of a side chain amino acid is a derivatised form selected from the group consisting of COOR, CONH 2 , CONHR and CONRR'.
  • the amino acid side chain is chemically protected, where appropriate.
  • the side chain protecting group may be a group as discussed below in relation to the group R L .
  • the present inventors have established that protected amino acid sequences are cleavable by enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
  • Lys Boc, Z-CI, Fmoc, Z, Alloc;
  • the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present.
  • the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group.
  • the amino acids selected are those having no reactive side chain functionality.
  • the amino acids may be selected from: Ala, Gly, lie, Leu, Met, Phe, Pro, and Val.
  • the dipeptide is used in combination with a self-immolative linker.
  • the self-immolative linker may be connected to -X 2 -.
  • -X 2 - is connected directly to the self-immolative linker.
  • the group -X 2 -CO- is connected to Y, where Y is NH, thereby forming the group -X2-CO-NH-.
  • A may comprise the functionality -CO- thereby to form an amide link with -X .
  • NH-X X 2 -CO-PABC- The PABC group is connected directly to the N10 position.
  • the self-immolative linker and the dipeptide together form the group -NH-Phe- Lys-CO-NH-PABC-, which is illustrated below:
  • the asterisk indicates the point of attachment to the N10 position
  • the wavy line indicates the point of attachment to the remaining portion of the linker L 1 or the point of attachment to A.
  • the wavy line indicates the point of attachment to A.
  • the side chain of the Lys amino acid may be protected, for example, with Boc, Fmoc, or Alloc, as described above.
  • the self-immolative linker and the dipeptide together form the group
  • A is a covalent bond.
  • L 1 and the antibody are directly connected.
  • L 1 comprises a contiguous amino acid sequence
  • the N-terminus of the sequence may connect directly to the antibody.
  • A is a covalent bond
  • the connection between the antibody and L 1 may be selected from:
  • An amino group of L 1 that connects to the antibody may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
  • An carboxyl group of L 1 that connects to the antibody may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
  • a hydroxyl group of L 1 that connects to the antibody may be derived from a hydroxyl group of an amino acid side chain, for example a serine amino acid side chain.
  • a thiol group of L 1 that connects to the antibody may be derived from a thiol group of an amino acid side chain, for example a serine amino acid side chain.
  • n 0 to 3
  • Y is a covalent bond or a functional group
  • E is an activatable group, for example by enzymatic action or light, thereby to generate a self-immolative unit.
  • the phenylene ring is optionally further substituted with one, two or three substituents as described herein.
  • the phenylene group is optionally further substituted with halo, N0 2 , R or OR.
  • n is 0 or 1 , most preferably 0.
  • E is selected such that the group is susceptible to activation, e.g. by light or by the action of an enzyme.
  • E may be -N0 2 or glucoronic acid.
  • the former may be susceptible to the action of a nitroreductase, the latter to the action of a ⁇ -glucoronidase.
  • E * is the activated form of E
  • Y is as described above.
  • the group Y may be a covalent bond to L 1 .
  • the group Y may be a functional group selected from
  • L 1 is a dipeptide
  • the dipeptide sequence need not be a substrate for an enzymatic activity.
  • A is a spacer group.
  • L 1 and the antibody are indirectly connected.
  • L 1 and A may be connected by a bond selected from:
  • the group A is:
  • n 0 to 6. In one embodiment, n is 5. In one embodiment, the group A is:
  • n is 0 to 6. In one embodiment, n is 5.
  • the group A is:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
  • m is 10 to 30, and preferably 20 to 30.
  • m is 0 to 50.
  • m is preferably 10-40 and n is 1 .
  • the group A is:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
  • m is 10 to 30, and preferably 20 to 30.
  • m is 0 to 50.
  • m is preferably 10-40 and n is 1 .
  • connection between the antibody and A is through a thiol residue of the antibody and a maleimide group of A. In one embodiment, the connection between the antibody and A is:
  • the S atom is typically derived from the antibody.
  • the maleimide-derived group is replaced with the group:
  • the maleimide-derived group is replaced with a group, which optionally together with the antibody, is selected from:
  • the maleimide-derived group is replaced with a group, which optionally together with the antibody, is selected from:
  • the wavy line indicates either the point of attachment to the antibody or the bond to the remaining portion of the A group
  • the asterisk indicates the other of the point of attachment to the antibody or the bond to the remaining portion of the A group.
  • the Connecting Group A is present, the Trigger L 1 is present and Self- Immolative Linker L 2 is absent.
  • L 1 and the Drug unit are directly connected via a bond.
  • L 2 is a bond. This may be particularly relevant when D L is of Formula II.
  • L 1 and D may be connected by a bond selected from:
  • L 1 and D are preferably connected by a bond selected from:
  • L 1 comprises a dipeptide and one end of the dipeptide is linked to D.
  • the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids.
  • the dipeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
  • the group -X X 2 - in dipeptide is selected from:
  • Cit is citrulline.
  • -NH- is the amino group of X-i
  • CO is the carbonyl group of X 2 .
  • the group -X X 2 - in dipeptide, -NH-XrX 2 -CO-, is selected from:
  • the group -X X 2 - in dipeptide, -NH-XrX 2 -CO-, is -Phe-Lys- or -Val-Ala-.
  • dipeptide combinations of interest include: -Gly-Gly-,
  • L 1 - D is: where -NH-XrX 2 -CO is the dipeptide, -N ⁇ is part of the Drug unit, the asterisk indicates the points of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L 1 or the point of attachment to A. Preferably, the wavy line indicates the point of attachment to A.
  • the dipeptide is valine-alanine and L 1 - D is:
  • the dipeptide is phenylalnine-lysine and L 1 - D is:
  • the dipeptide is valine-citrulline.
  • the groups A-L 1 are:
  • n 0 to 6. In one embodiment, n is 5. In one embodiment, the groups A-L 1 are:
  • n is 0 to 6. In one embodiment, n is 5.
  • the groups A-L 1 are:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the groups A-L 1 are:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7.
  • the groups A-L 1 are:
  • n is 0 to 6. In one embodiment, n is 5.
  • the groups A-L 1 are:
  • n is 0 to 6. In one embodiment, n is 5.
  • the groups A-L 1 are:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the groups A-L 1 is:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the groups A-L 1 are:
  • n 0 to 6. In one embodiment, n is 5.
  • the group A-L 1 are:
  • n 0 to 6. In one embodiment, n is 5.
  • the groups A 1 -L 1 are:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the groups A 1 are identical to each other. In one embodiment, the groups A 1
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8.
  • the groups A 1 -L 1 are:
  • n is 5.
  • n is 0 to 6. In one embodiment, n is 5.
  • the groups A 1 -L 1 are:
  • n is 0 or 1
  • m is 0 to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the groups A 1 are identical to each other. In one embodiment, the groups A 1
  • n is 0 or 1
  • m is to 30.
  • n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
  • the group R L is derivable from the group R L .
  • the group R L may be converted to a group R L by connection of an antibody to a functional group of R L .
  • Other steps may be taken to convert R L to R L . These steps may include the removal of protecting groups, where present, or the installation of an appropriate functional group.
  • Linkers can include protease-cleavable peptidic moieties comprising one or more amino acid units.
  • Peptide linker reagents may be prepared by solid phase or liquid phase synthesis methods (E. Schroder and K. Lubke, The Peptides, volume 1 , pp 76-136 (1965) Academic Press) that are well known in the field of peptide chemistry, including t-BOC chemistry (Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc/HBTU chemistry (Fields, G. and Noble, R.
  • Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine- phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly- val-cit) and glycine-glycine-glycine (gly-gly-gly)- Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • Amino acid side chains include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • the carbon atom to which the amino acid side chain is attached is chiral.
  • Each carbon atom to which the amino acid side chain is attached is independently in the (S) or (R) configuration, or a racemic mixture.
  • Drug-linker reagents may thus be enantiomerically pure, racemic, or
  • amino acid side chains are selected from those of natural and non-natural amino acids, including alanine, 2-amino-2-cyclohexylacetic acid, 2-amino-2- phenylacetic acid, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ⁇ -aminobutyric acid, ⁇ , ⁇ -dimethyl ⁇ - aminobutyric acid, ⁇ , ⁇ -dimethyl ⁇ -aminobutyric acid, ornithine, and citrulline (Cit).
  • alanine 2-amino-2-cyclohexylacetic acid
  • 2-amino-2- phenylacetic acid arginine, asparagine, aspartic acid
  • valine-citrulline (val-cit or vc) dipeptide linker reagent useful for constructing a linker-PBD drug moiety intermediate for conjugation to an antibody, having a para- aminobenzylcarbamoyl (PAB) self-immolative spacer has the structure:
  • Q is Ci-C 8 alkyl, -0-(Ci-C 8 alkyl), -halogen, -N0 2 or -CN; and m is an integer ranging from 0-4.
  • An exemplary phe-lys(Mtr) dipeptide linker reagent having a p-aminobenzyl group can be prepared according to Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60, and has the structure:
  • Mtr is mono-4-methoxytrityl
  • Q is Ci-C 8 alkyl, -0-(Ci-C 8 alkyl), -halogen, -N0 2 or -CN
  • m is an integer ranging from 0-4.
  • the "self-immolative linker” PAB para-aminobenzyloxycarbonyl
  • PAB para-aminobenzyloxycarbonyl
  • PAB self-immolative spacers besides PAB include, but are not limited to: (i) aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg.
  • Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4- aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem.
  • valine-citrulline dipeptide PAB analog reagent has a 2,6 dimethyl hen l rou and has the structure:
  • Linker reagents useful for the antibody drug conjugates of the invention include, but are not limited to: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo- SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, 1 ,8-bis- maleimidodiethyleneglycol (BM(PEO) 2 ), and 1 ,1 1-bis-maleimidotriethyleneglycol (BM(PEO) 3 ), which are commercially
  • Bis-maleimide reagents allow the attachment of a free thiol group of a cysteine residue of an antibody to a thiol-containing drug moiety, label, or linker intermediate, in a sequential or concurrent fashion.
  • Other functional groups besides maleimide, which are reactive with a thiol group of an antibody, PBD drug moiety, or linker intermediate include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.
  • linker reagents are: N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, Carlsson et al (1978) Biochem. J.
  • succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate SMCC
  • iminothiolane IT
  • bifunctional derivatives of imidoesters such as dimethyl adipimidate HCI
  • active esters such as disuccinimidyl suberate
  • aldehydes such as glutaraldehyde
  • bis- azido compounds such as bis (p-azidobenzoyl) hexanediamine
  • bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine
  • diisocyanates such as toluene 2,6- diisocyanate
  • bis-active fluorine compounds such as 1 ,5-difluoro-2,4-dinitrobenzene.
  • Useful linker reagents can also be obtained via other commercial sources, such as
  • the Linker may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to an antibody (US 2006/1 16422; US 2005/271615; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc.
  • Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which is related to the potency of the ADC.
  • loading i.e. loading
  • a multitude of drug moieties may be attached through a dendritic or branched linker.
  • the conjugate of the first aspect of the invention may have a capping group R c at the N10 position.
  • Compound E may have a capping group R c .
  • the group R 10 in one of the monomer units is a capping group R c or is a group R 10 .
  • the group R 10 in one of the monomer units is a capping group R c .
  • the group R L in one of the monomer units is a capping group R c or is a linker for connection to an antibody. In one embodiment, where compound E is a dimer with each monomer being of formula (E), the group R L in one of the monomer units is a capping group R c .
  • the group R c is removable from the N10 position of the PBD moiety to leave an N 10-C1 1 imine bond, a carbinolamine, a substituted carbinolamine, where QR 11 is OS0 3 M, a bisulfite adduct, a thiocarbinolamine, a substituted thiocarbinolamine, or a substituted carbinalamine.
  • R c may be a protecting group that is removable to leave an N10-C1 1 imine bond, a carbinolamine, a substituted cabinolamine, or, where QR 11 is OS0 3 M, a bisulfite adduct. In one embodiment, R c is a protecting group that is removable to leave an N10-C1 1 imine bond.
  • the group R c is intended to be removable under the same conditions as those required for the removal of the group R 10 , for example to yield an N10-C1 1 imine bond, a carbinolamine and so on.
  • the capping group acts as a protecting group for the intended functionality at the N10 position.
  • the capping group is intended not to be reactive towards an antibody.
  • R c is not the same as R L .
  • the capping group may be used as intermediates in the synthesis of dimers having an imine monomer.
  • compounds having a capping group may be used as conjugates, where the capping group is removed at the target location to yield an imine, a carbinolamine, a substituted cabinolamine and so on.
  • the capping group may be referred to as a therapeutically removable nitrogen protecting group, as defined in the inventors' earlier application WO 00/12507.
  • the group R c is removable under the conditions that cleave the linker R L of the group R 10 .
  • the capping group is cleavable by the action of an enzyme.
  • the capping group is removable prior to the connection of the linker R L to the antibody.
  • the capping group is removable under conditions that do not cleave the linker R L .
  • the capping group is removable prior to the addition or unmasking of G 1 .
  • the capping group may be used as part of a protecting group strategy to ensure that only one of the monomer units in a dimer is connected to an antibody.
  • the capping group may be used as a mask for a N10-C1 1 imine bond.
  • the capping group may be removed at such time as the imine functionality is required in the compound.
  • the capping group is also a mask for a carbinolamine, a substituted cabinolamine, and a bisulfite adduct, as described above.
  • R c may be an N10 protecting group, such as those groups described in the inventors' earlier application, WO 00/12507. In one embodiment, R c is a therapeutically removable nitrogen protecting group, as defined in the inventors' earlier application, WO 00/12507.
  • R c is a carbamate protecting group.
  • the carbamate protecting group is selected from:
  • the carbamate protecting group is further selected from Moc.
  • R c is a linker group R L lacking the functional group for connection to the antibody.
  • This application is particularly concerned with those R c groups which are carbamates.
  • L 1 is as defined above in relation to R 10 .
  • L 2 is as defined above in relation to R 10 .
  • G 2 is Ac (acetyl) or Moc, or a carbamate protecting group selected from:
  • the carbamate protecting group is further selected from Moc.
  • the acyl group together with an amino group of L 3 or L 2 may form an amide bond.
  • the acyl group together with a hydroxy group of L 3 or L 2 may form an ester bond.
  • G 3 is heteroalkyi.
  • the heteroalkyi group may comprise polyethylene glycol.
  • the heteroalkyi group may have a heteroatom, such as O or N, adjacent to the acyl group, thereby forming a carbamate or carbonate group, where appropriate, with a heteroatom present in the group L 3 or L 2 , where appropriate.
  • G 3 is selected from NH 2 , NHR and NRR'.
  • G 3 is NRR'.
  • G 2 is the group: where the asterisk indicates the point of attachment to L 3
  • n is 0 to 6
  • G 4 is selected from OH, OR, SH, SR, COOR, CONH 2 , CONHR, CONRR', NH 2 , NHR, NRR', N0 2 , and halo.
  • the groups OH, SH, NH 2 and NHR are protected.
  • n is 1 to 6, and preferably n is 5.
  • G 4 is OR, SR, COOR, CONH 2 , CONHR,
  • G 4 is OR, SR, and NRR'.
  • G 4 is selected from OR and NRR', most preferably G 4 is OR.
  • Most preferably G 4 is OMe.
  • the group G 2 is:
  • the group G 2 is:
  • n is 0 or 1
  • m is 0 to 50
  • G 4 is selected from OH, OR, SH, SR, COOR, CONH 2 , CONHR, CONRR', NH 2 , NHR, NRR', N0 2 , and halo.
  • n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
  • n is 1 and m is 10 to 50, preferably 20 to 40.
  • the groups OH, SH, NH 2 and NHR are protected.
  • G 4 is OR, SR, COOR, CONH 2 , CONHR, CONRR', and NRR'.
  • G 4 is OR, SR, and NRR'.
  • G 4 is selected from OR and NRR', most preferably G 4 is OR.
  • G 4 is OMe.
  • the group G 2 is:
  • the group G 2 is:
  • n is 1-20, m is 0-6, and G 4 is selected from OH, OR, SH, SR, COOR, CONH 2 , CONHR, CONRR', NH 2 , NHR, NRR', N0 2 , and halo.
  • n is 1 -10.
  • n is 10 to 50, preferably 20 to 40.
  • n is 1 .
  • m is 1.
  • the groups OH, SH, NH 2 and NHR are protected.
  • G 4 is OR, SR, COOR, CONH 2 , CONHR, CONRR', and NRR'.
  • G 4 is OR, SR, and NRR'.
  • G 4 is selected from OR and NRR', most preferably G 4 is OR.
  • G 4 is OMe.
  • the group G 2 is:
  • n, m and G 4 are as defined above.
  • G 4 may be OH, SH, NH 2 and NHR. These groups are preferably protected.
  • OH is protected with Bzl, TBDMS, or TBDPS.
  • SH is protected with Acm, Bzl, Bzl-OMe, Bzl-Me, or Trt.
  • NH 2 or NHR are protected with Boc, Moc, Z-CI, Fmoc, Z, or Alloc.
  • the group G 2 is present in combination with a group L 3 , which group is a dipeptide.
  • the capping group is not intended for connection to the antibody.
  • the other monomer present in the dimer serves as the point of connection to the antibody via a linker.
  • the functionality present in the capping group is not available for reaction with an antibody.
  • reactive functional groups such as OH, SH, NH 2 , COOH are preferably avoided.
  • such functionality may be present in the capping group if protected, as described above.
  • Embodiments of the present invention include ConjA wherein the antibody is as defined above.
  • Embodiments of the present invention include ConjB wherein the antibody is as defined above.
  • Embodiments of the present invention include ConjC wherein the antibody is as defined above.
  • Embodiments of the present invention include ConjD wherein the antibody is as defined above.
  • Embodiments of the present invention include ConjE wherein the antibody is as defined above.
  • some embodiments of the invention exclude ConjA, ConjB, ConjC, ConjD and ConjE.
  • the drug loading is the average number of PBD drugs per antibody, e.g. antibody.
  • drug loading may range from 1 to 8 drugs (D L ) per antibody, i.e. where 1 , 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the antibody.
  • Compositions of conjgates include collections of antibodies, conjugated with a range of drugs, from 1 to 8.
  • drug loading may range from 1 to 80 drugs (D L ) per antibody, although an upper limit of 40, 20, 10 or 8 may be preferred.
  • Compositions of conjgates include collections of antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.
  • the average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
  • the quantitative distribution of ADC in terms of p may also be determined.
  • ELISA the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 1 1 :843-852).
  • the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA.
  • ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues.
  • separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • Such techniques are also applicable to other types of conjugates.
  • p may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • Higher drug loading, e.g. p >5 may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
  • an antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety.
  • cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions.
  • DTT dithiothreitol
  • the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
  • Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 1 14(13):2721 -2729; US 7521541 ; US 7723485; WO2009/052249).
  • the engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties.
  • the location of the drug moiety can thus be designed, controlled, and known.
  • the drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield.
  • Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody.
  • a drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.
  • site-specific conjugation can be achieved by engineering antibodies to contain unnatural amino acids in their heavy and/or light chains as described by Axup et al. ((2012), Proc Natl Acad Sci U S A. 109(40):16101 -161 16).
  • the unnatural amino acids provide the additional advantage that orthogonal chemistry can be designed to attach the linker reagent and drug.
  • the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1 , 2, 3, etc.
  • Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value.
  • Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.
  • the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
  • the average number of dimer pyrrolobenzodiazepine groups per antibody is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.
  • dimer pyrrolobenzodiazepine group per antibody.
  • a reference to carboxylic acid also includes the anionic (carboxylate) form (-COO " ), a salt or solvate thereof, as well as conventional protected forms.
  • a reference to an amino group includes the protonated form (-N + HR 1 R 2 ), a salt or solvate of the amino group, for example, a
  • hydrochloride salt as well as conventional protected forms of an amino group.
  • a reference to a hydroxyl group also includes the anionic form (-0 " ), a salt or solvate thereof, as well as conventional protected forms.
  • a corresponding salt of the active compound for example, a pharmaceutically-acceptable salt.
  • a pharmaceutically-acceptable salt examples are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as ⁇ 3 .
  • suitable organic cations include, but are not limited to, ammonium ion (i.e. NH 4 + ) and substituted ammonium ions (e.g. NH 3 R + , NH 2 R2 + , NHR 3 + , NR 4 + ).
  • suitable substituted ammonium ions are those derived from: ethylamine, diethylamine,
  • a common quaternary ammonium ion is N(CH 3 ) 4 + .
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids:
  • hydrochloric hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,
  • Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
  • solvate is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • the invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R A OH, where R A is Ci-4 alkyl):
  • carbinolamine and carbinolamine ether forms of the PBD can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R 10 above).
  • the balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
  • Certain compounds of the invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or "isomeric forms").
  • chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • stereoisomers refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
  • Enantiomers refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • the compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention.
  • a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
  • the terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • isomers are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space).
  • a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH.
  • a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl.
  • Ci -7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para- methoxyphenyl).
  • keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
  • tautomer or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers include interconversions by reorganization of some of the bonding electrons.
  • H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 0 and 18 0; and the like.
  • isotopes examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to 2 H (deuterium, D), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 CI, and 125 l.
  • isotopically labeled compounds of the present invention for example those into which radioactive isotopes such as 3H, 13C, and 14C are incorporated.
  • Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single- photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
  • An 18F labeled compound may be useful for PET or SPECT studies.
  • Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • substitution with heavier isotopes, particularly deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent.
  • the concentration of such a heavier isotope, specifically deuterium may be defined by an isotopic enrichment factor.
  • any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
  • a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
  • Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
  • the cytotoxic or cytostatic activity of an antibody-drug conjugate is measured by: exposing mammalian cells having receptor proteins to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability.
  • Cell-based in vitro assays are used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of an ADC of the invention.
  • the in vitro potency of antibody-drug conjugates can be measured by a cell proliferation assay.
  • the CellTiter-Glo ® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, Wl), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (US Patent Nos. 5583024; 5674713 and 5700670). This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol. Meth. 160:81 -88; US 6602677).
  • the CellTiter-Glo ® Assay is conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree et al (1995) Anticancer Drugs 6:398-404).
  • the homogeneous assay procedure involves adding the single reagent (CellTiter-Glo ® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required.
  • the system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing.
  • the cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.
  • the homogeneous "add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • the CellTiter-Glo ® Assay generates a "glow-type" luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on cell type and medium used. Viable cells are reflected in relative luminescence units (RLU).
  • the substrate, Beetle Luciferin is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons.
  • the in vitro potency of antibody-drug conjugates can also be measured by a cytotoxicity assay.
  • Cultured adherent cells are washed with PBS, detached with trypsin, diluted in complete medium, containing 10% FCS, centrifuged, re-suspended in fresh medium and counted with a haemocytometer. Suspension cultures are counted directly. Monodisperse cell suspensions suitable for counting may require agitation of the suspension by repeated aspiration to break up cell clumps.
  • the cell suspension is diluted to the desired seeding density and dispensed (1 ⁇ per well) into black 96 well plates. Plates of adherent cell lines are incubated overnight to allow adherence. Suspension cell cultures can be used on the day of seeding.
  • a stock solution (1 ml) of ADC (20pg/ml) is made in the appropriate cell culture medium.
  • Serial 10-fold dilutions of stock ADC are made in 15ml centrifuge tubes by serially transferring 10 ⁇ to 900 ⁇ of cell culture medium.
  • Alamar blue assay At the end of the incubation period, cell viability is assessed with the Alamar blue assay.
  • AlamarBlue Invitrogen
  • Alamar blue fluorescence is measured at excitation 570nm, emission 585nm on the Varioskan flash plate reader. Percentage cell survival is calculated from the mean fluorescence in the ADC treated wells compared to the mean fluorescence in the control wells.
  • the conjugates of the invention may be used to provide a PBD compound at a target location.
  • the target location is preferably a proliferative cell population.
  • the antibody is an antibody for an antigen present on a proliferative cell population.
  • the antigen is absent or present at a reduced level in a non-proliferative cell population compared to the amount of antigen present in the proliferative cell population, for example a tumour cell population.
  • the linker may be cleaved so as to release a compound RelA, RelB, ReIC, RelD or RelE.
  • the conjugate may be used to selectively provide a compound RelA, RelB, Rel C, RelD or RelE to the target location.
  • the linker may be cleaved by an enzyme present at the target location.
  • the target location may be in vitro, in vivo or ex vivo.
  • the antibody-drug conjugate (ADC) compounds of the invention include those with utility for anticancer activity.
  • the compounds include an antibody conjugated, i.e.
  • ADC antibody-drug conjugates
  • the present invention provides a conjugate compound as described herein for use in therapy.
  • conjugate compound as described herein for use in the treatment of a proliferative disease.
  • a second aspect of the present invention provides the use of a conjugate compound in the manufacture of a medicament for treating a proliferative disease.
  • proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g.
  • lung cancer small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
  • Cancers of particular interest include, but are not limited to, leukemias and ovarian cancers.
  • Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
  • gastrointestinal including, e.g. bowel, colon
  • breast mammary
  • ovarian prostate
  • liver hepatic
  • kidney renal
  • bladder pancreas
  • brain and skin.
  • non-Hodgkin Lymphoma including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphatic lymphoma (CLL) nd leukemias such as Hairy cell leukemia (HCL), Hairy cell leukemia variant (HCL-v) and Acute Lymphoblastic Leukaemia (ALL).
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • MCL Mantle Cell lymphoma
  • CLL chronic lymphatic lymphoma
  • HCL Hairy cell leukemia
  • HCL-v Hairy cell leukemia variant
  • ALL Acute Lymphoblastic Leukaemia
  • the antibody-drug conjugates (ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen.
  • exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, haematological, and lymphoid malignancies.
  • Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic, including autoimmune, disorders.
  • the disease or disorder to be treated is a hyperproliferative disease such as cancer.
  • cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer,
  • adenocarcinoma of the lung and squamous carcinoma of the lung cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • Autoimmune diseases for which the ADC compounds may be used in treatment include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g.
  • autoimmune gastritis and pernicious anemia such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis
  • vasculitis such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteritis
  • autoimmune neurological disorders such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies
  • renal disorders such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease
  • autoimmune dermatologic disorders such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid,
  • Graves' disease and thyroiditis More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and
  • the conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention.
  • a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention.
  • terapéuticaally effective amount is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • a compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, such as chemotherapeutics); surgery; and radiation therapy.
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids,
  • Chemotherapeutic agents include compounds used in "targeted therapy” and conventional chemotherapy.
  • chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(ll), CAS No. 15663-27-1 ), carboplatin (CAS No.
  • paclitaxel TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.
  • trastuzumab HERCEPTIN®, Genentech
  • temozolomide 4-methyl-5-oxo- 2,3,4,6,8- pentazabicyclo [4.3.0] nona-2,7,9-triene- 9-carboxamide, CAS No. 85622-93-1 ,
  • TEMODAR® TEMODAL®, Schering Plough
  • tamoxifen (Z)-2-[4-(1 ,2-diphenylbut-1- enyl)phenoxy]-/V,/V-dimethylethanamine
  • NOLVADEX® NOLVADEX®
  • ISTUBAL® ISTUBAL®
  • VALODEX® doxorubicin
  • ADRIAMYCIN® doxorubicin
  • chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU1 1248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1 126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (si
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolast
  • calicheamicin calicheamicin gammal l, calicheamicin omegaH (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an
  • esperamicin as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
  • cyanomorpholino-doxorubicin 2-pyrrolino-doxorubicin and deoxydoxorubicin
  • epirubicin esorubicin, idarubicin, nemorubicin, marcellomycin
  • mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin
  • anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-aza
  • etoglucid gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
  • pentostatin phenamet
  • pirarubicin losoxantrone
  • podophyllinic acid 2-ethylhydrazide
  • PSK® polysaccharide complex JHS Natural Products, Eugene, OR
  • razoxane rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
  • pipobroman gacytosine; arabinoside ("Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin;
  • vinblastine etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine
  • NAVELBINE® novantrone
  • teniposide edatrexate
  • daunomycin aminopterin
  • capecitabine XELODA®, Roche
  • ibandronate CPT-1 1 ; topoisomerase inhibitor RFS 2000;
  • DMFO difluoromethylornithine
  • retinoids such as retinoic acid
  • pharmaceutically acceptable salts, acids and derivatives of any of the above DMFO
  • DMFO difluoromethylornithine
  • chemotherapeutic agent include: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole
  • panitumumab VECTIBIX®, Amgen
  • rituximab RVUXAN®, Genentech/Biogen personalize
  • ARZERRA® GSK
  • pertuzumab PERJETATM, OMNITARGTM, 2C4, Genentech
  • trastuzumab HERCEPTIN®, Genentech
  • tositumomab Bexxar, Corixia
  • antibody drug conjugate gemtuzumab ozogamicin
  • Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
  • tacatuzumab tetraxetan tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.
  • compositions according to the present invention may comprise, in addition to the active ingredient, i.e. a conjugate compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. a conjugate compound
  • carrier e.g. a pharmaceutically acceptable excipient
  • buffer e.g. cutaneous, subcutaneous, or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • a capsule may comprise a solid carrier such a gelatin.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • conjugate compound While it is possible for the conjugate compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.
  • the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutical composition e.g., formulation, preparation, medicament
  • a pharmaceutically acceptable carrier e.g., diluent, or excipient.
  • the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other
  • compositions further comprises other active agents, for example, other therapeutic or prophylactic agents.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and
  • Another aspect of the present invention pertains to methods of making a pharmaceutical composition
  • a pharmaceutical composition comprising admixing at least one [ 11 C]-radiolabelled conjugate or conjugate-like compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
  • pharmaceutically acceptable as used herein, pertains to compounds,
  • ingredients, materials, compositions, dosage forms, etc. which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
  • the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
  • the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
  • sterile liquids e.g., solutions, suspensions
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers,
  • bacteriostats suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 ⁇ g ml, for example from about 10 ng/ml to about 1 ⁇ g/ml.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
  • the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
  • a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 ⁇ g to about 10 mg) per kilogram body weight of the subject per day.
  • the active compound is a salt, an ester, an amide, a prodrug, or the like
  • the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
  • the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily. In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.
  • the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.
  • the conjugate compound is administered to a human patient according to the following dosage regime: about 50 or about 75 mg, 3 or 4 times daily. In one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 100 or about 125 mg, 2 times daily.
  • the dosage amounts described above may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of PBD compound provided, for example the amount of compound that is releasable after cleavage of the linker.
  • an ADC of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the molecule is suitably
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
  • An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Treatment
  • treatment pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis, prevention is also included.
  • terapéuticaally-effective amount pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • prophylactically-effective amount refers to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • Antibody drug conjugates may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including reaction of a nucleophilic group of an antibody with a drug-linker reagent. This method may be employed to prepare the antibody-drug conjugates of the invention.
  • Nucleophilic groups on antibodies include, but are not limited to side chain thiol groups, e.g. cysteine.
  • Thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties such as those of the present invention.
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem.
  • Each cysteine disulfide bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • the subject/patient may be an animal, mammal, a placental mammal, a marsupial
  • a monotreme e.g., duckbilled platypus
  • a rodent e.g., a guinea pig, a hamster, a rat, a mouse
  • murine e.g., a mouse
  • a lagomorph e.g., a rabbit
  • avian e.g., a bird
  • canine e.g., a dog
  • feline e.g., a cat
  • equine e.g., a horse
  • porcine e.g., a pig
  • ovine e.g., a sheep
  • bovine e.g., a cow
  • a primate simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a
  • the subject/patient may be any of its forms of development, for example, a foetus.
  • the subject/patient is a human.
  • R 6 , R 7 , R 9 , and Y' are preferably the same as R 6 , R 7 , R 9 , and Y respectively.
  • Y and Y' are preferably O.
  • R" is preferably a C 3-7 alkylene group with no substituents. More preferably R" is a C 3 , C 5 or C 7 alkylene. Most preferably, R" is a C 3 or C 5 alkylene. R 6 to R 9
  • R 9 is preferably H.
  • R 6 is preferably selected from H, OH, OR, SH, NH 2 , nitro and halo, and is more preferably H or halo, and most preferably is H.
  • R 7 is preferably selected from H, OH, OR, SH, SR, NH 2 , NHR, NRR', and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted Ci -7 alkyl, C 3- io heterocyclyl and C 5- io aryl groups. R may be more preferably a Ci -4 alkyl group, which may or may not be substituted.
  • a substituent of interest is a C 5 -6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7- positions are OMe and OCH 2 Ph. Other substituents of particular interest are dimethylamino (i.e.
  • R 12 is selected from:
  • each of R , R and R are independently selected from H, Ci -3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon ato group is no more than 5;
  • R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy;
  • R 24 is selected from: H; C 1-3 saturated alkyl; C 2- 3 alkenyl; C 2- 3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
  • R 12 When R 12 is a C5-10 aryl group, it may be a C5-7 aryl group.
  • a C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl.
  • R 12 is preferably phenyl.
  • R 12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
  • R 12 When R 12 is a C5-10 aryl group, it may be a C 8- io aryl, for example a quinolinyl or isoquinolinyl group.
  • the quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position.
  • the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred.
  • the isoquinolinyl may be isoquinolin-1 -yl, isoquinolin-3- yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
  • R 12 When R 12 is a C 5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred.
  • the substituents may be any position.
  • R 12 is C 5 - 7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or ⁇ to the bond to the remainder of the compound. Therefore, where the C 5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
  • R 12 is a C 8- io aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
  • R 12 substituents, when R 12 is a C 5 -w aryl group
  • R 12 when R 12 is a C 5-10 aryl group is halo, it is preferably F or CI, more preferably CI.
  • R 12 when R 12 is a C 5- io aryl group is ether, it may in some embodiments be an alkoxy group, for example, a Ci -7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C 5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy).
  • the alkoxy group may itself be further substituted, for example by an amino group (e.g.
  • a substituent on R 12 when R 12 is a C 5- io aryl group is Ci -7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
  • a substituent on R 12 when R 12 is a C5-10 aryl group is C 3-7 heterocyclyl, it may in some embodiments be C 6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C 1-4 alkyl groups. If the C 6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
  • substituent on R 12 when R 12 is a C 5-10 aryl group is bis-oxy-C 1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene. If a substituent on R 12 when R 12 is a C5-10 aryl group is ester, this is preferably methyl ester or ethyl ester. Particularly preferred substituents when R 12 is a C 5- io aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl. Other particularly preferred substituent for R 12 are dimethylaminopropyloxy and carboxy.
  • Particularly preferred substituted R 12 groups when R 12 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.
  • R 12 groups of particular interest include 4-(4- methylpiperazin-1 -yl)phenyl and 3,4-bisoxymethylene-phenyl.
  • R 12 When R 12 is Ci -5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched. When R 12 is C 3- 6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
  • each of R , R and R are independently selected from H, Ci -3 saturated alkyl, C 2-3 alkenyl, C 2- 3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 12 group is no more than 5. In some embodiments, the total number of carbon atoms in the R 12 group is no more than 4 or no more than 3.
  • one of R 21 , R 22 and R 23 is H , with the other two groups being selected from H, Ci-3 saturated alkyl, C 2- 3 alkenyl, C 2- 3 alkynyl and cyclopropyl.
  • two of R 21 , R 22 and R 23 are H, with the other group being selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl.
  • the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
  • R is H.
  • R is H.
  • R is H.
  • R and R are H
  • R and R are H.
  • R and R are H
  • R 12 group of particular interest is:
  • R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy;
  • the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
  • R 24 is selected from: H; d -3 saturated alkyl; C 2- 3 alkenyl; C 2- 3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
  • R 24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R 24 is selected from H and methyl.
  • R and R are independently selected from H, F, C1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C 1-4 alkyl amido and C 1-4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C 1-4 alkyl ester.
  • R 26a and R 26b are both H.
  • R 26a and R 26b are both methyl.
  • one of R 26a and R 26b is H, and the other is selected from Ci -4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
  • the group which is not H is selected from methyl and ethyl.
  • R is of formula lla.
  • a in R when it is of formula I la may be phenyl group or a C 5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl.
  • Q 2 -X may be on any of the available ring atoms of the C 5-7 aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or Y to the bond to the remainder of the compound. Therefore, where the C 5-7 aryl group (A) is phenyl, the substituent (Q 2 -X) is preferably in the meta- or para- positions, and more preferably is in the para- position.
  • Q 1 is a single bond.
  • Q 2 is selected from a single bond and -Z-(CH 2 ) n -, where Z is selected from a single bond, O, S and NH and is from 1 to 3.
  • Q 2 is a single bond.
  • Q 2 is -Z- (CH 2 ) n -.
  • Z may be O or S and n may be 1 or n may be 2.
  • Z may be a single bond and n may be 1.
  • R is of formula lib.
  • R , R and R are independently selected from H and unsubstituted Ci -2 alkyl. In some preferred
  • R C1 , R C2 and R C3 are all H. In other embodiments, R C1 , R C2 and R C3 are all methyl. In certain embodiments, R C1 , R C2 and R C3 are independently selected from H and methyl.
  • R L2 NHNH-R L2 , CONHNH-R L2 , , NR N R L2' , wherein R N is selected from the group comprising H and C 1 -4 alkyl.
  • X may preferably be: OH, SH, C0 2 H,
  • PPaarrttiiccuullaarrllyy pprreeff(erred groups include: 0-R L2' , S-R L2' and NH-R L2' , with NH-R L2' being the most preferred group.
  • R is of formula lie. In these embodiments, it is preferred that Q is NR N -R L2' . In other embodiments, Q is 0-R L2' . In further embodiments, Q is S-R L2' . R N is preferably selected from H and methyl. In some embodiment, R N is H. In other words,
  • R N is methyl
  • R 22 may be -A-CH 2 -X and -A-X.
  • X may be O- R L2' , S-R L2' , C0 2 -R L2' , CO-R L2' and NH-R L2' .
  • X may be NH-R L2' .
  • R 10 and R 11 together form a double bond between the nitrogen and carbon atoms to which they are bound.
  • R 11 is OH
  • R 11 is OMe. In some embodiments, R 11 is SO z M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation.
  • R 11a is OH. In some embodiments, R 11a is OMe.
  • R 11a is SO z M, where z is 2 or 3 and M is a monovalent
  • R 20 and R 21 together form a double bond between the nitrogen and carbon atoms to which they are bound.
  • R 20 is H.
  • R 20 is R c .
  • R 21 is OH.
  • R 21 is OMe.
  • R is SO z M, where z is 2 or 3 and M is a monovalent
  • R 30 and R 31 together form a double bond between the nitrogen and carbon atoms to which they are bound.
  • R 31 is OH
  • R is OMe
  • R is SO z M, where z is 2 or 3 and M is a monovalent
  • M is a monovalent pharmaceutically acceptable cation, and is more preferably Na + .
  • z is preferably 3.
  • Preferred conjugates of the first aspect of the present invention may have a D L of formula la:
  • R L , R 20 and R 21 are as defined above;
  • n 1 or 3;
  • R 1a is methyl or phenyl
  • R 2a is selected from: (b) c)
  • Preferred conjugates of the first aspect of the present invention may have a D L of formula lb:
  • R L , R 20 and R 21 are as defined above;
  • n 1 or 3;
  • R 1a is methyl or phenyl.
  • Preferred conjugates of the first aspect of the present invention may have a D L of formula Ic:
  • R L2' , R 10 , R 11 , R 30 and R 31 are as defined above
  • n 1 or 3;
  • R 12a is selected from:
  • the amino group is at either the meta or para positions of the phenyl group.
  • Preferred conjugates of the first aspect of the present invention may have a D L of formula Id
  • R L2' , R 10 , R 11 , R 30 and R 31 are as defined above
  • n 1 or 3;
  • R 1a is methyl or phenyl
  • R 12a is selected from:
  • Preferred conjugates of the first aspect of the present invention may have a D L of formula le:
  • R , R , R JU and R J1 are as defined above
  • n 1 or 3;
  • R 1a is methyl or phenyl
  • R 12a is selected from:
  • MS Mass spectroscopy
  • Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from

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Abstract

L'invention concerne des conjugués d'un anticorps anti-CD22 humanisé isolé et de dimères PBD.
PCT/EP2013/071347 2012-10-12 2013-10-11 Conjugués anticorps anti-cd22 - pyrrolobenzodiazépine WO2014057118A1 (fr)

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US201361794843P 2013-03-15 2013-03-15
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Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015196089A1 (fr) * 2014-06-20 2015-12-23 Bioalliance C.V. Conjugués anticorps anti-cd22-médicament et leurs méthodes d'utilisation
EP2841459A4 (fr) * 2012-04-26 2016-04-13 Bioatla Llc Anticorps anti-cd22
US9387259B2 (en) 2011-10-14 2016-07-12 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9388187B2 (en) 2011-10-14 2016-07-12 Medimmune Limited Pyrrolobenzodiazepines
US9399073B2 (en) 2011-10-14 2016-07-26 Seattle Genetics, Inc. Pyrrolobenzodiazepines
US9399641B2 (en) 2011-09-20 2016-07-26 Medimmune Limited Pyrrolobenzodiazepines as unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates
US9408923B2 (en) 2012-12-21 2016-08-09 Bioalliance C.V. Hydrophilic self-immolative linkers and conjugates thereof
US9415117B2 (en) 2012-10-12 2016-08-16 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016166301A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués de médicament-anticorps spécifiques de site
WO2016166307A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués anticorps-médicaments spécifiques de sites
US9526798B2 (en) 2011-10-14 2016-12-27 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9562049B2 (en) 2012-12-21 2017-02-07 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9567340B2 (en) 2012-12-21 2017-02-14 Medimmune Limited Unsymmetrical pyrrolobenzodiazepines-dimers for use in the treatment of proliferative and autoimmune diseases
US9592240B2 (en) 2010-04-15 2017-03-14 Seattle Genetics Inc. Targeted pyrrolobenzodiazapine conjugates
US9624227B2 (en) 2008-10-17 2017-04-18 Medimmune Limited Unsymmetrical pyrrolobenzodiazepine-dimers for treatment of proliferative diseases
US9649390B2 (en) 2013-03-13 2017-05-16 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9732084B2 (en) 2010-04-15 2017-08-15 Medimmune Limited Pyrrolobenzodiazepines used to treat proliferative diseases
US9745303B2 (en) 2012-10-12 2017-08-29 Medimmune Limited Synthesis and intermediates of pyrrolobenzodiazepine derivatives for conjugation
EP2906251B1 (fr) * 2012-10-12 2017-09-27 ADC Therapeutics SA Conjugués anticorps anti-cd22 - pyrrolobenzodiazépine
US9821074B2 (en) 2013-03-13 2017-11-21 Genentech, Inc. Pyrrolobenzodiazepines and conjugates thereof
US9889207B2 (en) 2012-10-12 2018-02-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9950077B2 (en) 2014-06-20 2018-04-24 Bioalliance C.V. Anti-folate receptor alpha (FRA) antibody-drug conjugates and methods of using thereof
US9956298B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10058613B2 (en) 2015-10-02 2018-08-28 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
US10179820B2 (en) 2014-09-12 2019-01-15 Genentech, Inc. Anti-HER2 antibodies and immunoconjugates
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10472422B2 (en) 2016-01-08 2019-11-12 Abgenomics International Inc. Tetravalent anti-PSGL-1 antibodies and uses thereof
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11384098B2 (en) 2017-02-08 2022-07-12 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11426467B2 (en) 2017-06-14 2022-08-30 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD25 ADC
US11484606B2 (en) 2019-06-07 2022-11-01 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
US11583590B2 (en) 2017-09-29 2023-02-21 Daiichi Sankyo Company, Limited Antibody-pyrrolobenzodiazepine derivative conjugate and method of use thereof for treating a tumor
US11596696B2 (en) 2017-04-20 2023-03-07 Adc Therapeutics Sa Combination therapy with an anti-CD25 antibody-drug conjugate
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11752197B2 (en) 2019-08-12 2023-09-12 Regeneron Pharmaceuticals, Inc. Macrophage stimulating 1 receptor (MST1R) variants and uses thereof
US11976122B2 (en) 2020-07-31 2024-05-07 Adc Therapeutics Sa Anti-IL13Rα2 antibodies

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003104425A2 (fr) * 2002-06-07 2003-12-18 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Nouveaux anticorps anti-cd22 stables
EP1609803A1 (fr) * 2003-03-31 2005-12-28 Chugai Seiyaku Kabushiki Kaisha Anticorps modifie dirige contre cd22 et son utilisation
WO2011050262A2 (fr) * 2009-10-23 2011-04-28 Abbott Laboratories Immunoglobulines à domaine variable double et utilisations de celles-ci
WO2011130613A1 (fr) * 2010-04-15 2011-10-20 Seattle Genetics, Inc. Conjugués de pyrrolobenzodiazapine ciblés
WO2011130616A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines utilisées pour traiter des maladies prolifératives
WO2011130598A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines et conjugués de celles-ci
WO2012112687A1 (fr) * 2011-02-15 2012-08-23 Immunogen, Inc. Procédés de préparation de conjugués
WO2013055987A1 (fr) * 2011-10-14 2013-04-18 Spirogen Sàrl Pyrrolobenzodiazépines et conjugués associés
WO2013163519A1 (fr) * 2012-04-26 2013-10-31 Jay Short Anticorps anti-cd22
WO2013177481A1 (fr) * 2012-05-25 2013-11-28 Immunogen, Inc. Benzodiazépines et leurs conjugués
WO2014011518A1 (fr) * 2012-07-09 2014-01-16 Genentech, Inc. Immunoconjugués comprenant des anticorps anti-cd22

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003104425A2 (fr) * 2002-06-07 2003-12-18 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Nouveaux anticorps anti-cd22 stables
EP1609803A1 (fr) * 2003-03-31 2005-12-28 Chugai Seiyaku Kabushiki Kaisha Anticorps modifie dirige contre cd22 et son utilisation
WO2011050262A2 (fr) * 2009-10-23 2011-04-28 Abbott Laboratories Immunoglobulines à domaine variable double et utilisations de celles-ci
WO2011130613A1 (fr) * 2010-04-15 2011-10-20 Seattle Genetics, Inc. Conjugués de pyrrolobenzodiazapine ciblés
WO2011130616A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines utilisées pour traiter des maladies prolifératives
WO2011130598A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines et conjugués de celles-ci
WO2012112687A1 (fr) * 2011-02-15 2012-08-23 Immunogen, Inc. Procédés de préparation de conjugués
WO2013055987A1 (fr) * 2011-10-14 2013-04-18 Spirogen Sàrl Pyrrolobenzodiazépines et conjugués associés
WO2013163519A1 (fr) * 2012-04-26 2013-10-31 Jay Short Anticorps anti-cd22
WO2013177481A1 (fr) * 2012-05-25 2013-11-28 Immunogen, Inc. Benzodiazépines et leurs conjugués
WO2014011518A1 (fr) * 2012-07-09 2014-01-16 Genentech, Inc. Immunoconjugués comprenant des anticorps anti-cd22

Cited By (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9624227B2 (en) 2008-10-17 2017-04-18 Medimmune Limited Unsymmetrical pyrrolobenzodiazepine-dimers for treatment of proliferative diseases
US9592240B2 (en) 2010-04-15 2017-03-14 Seattle Genetics Inc. Targeted pyrrolobenzodiazapine conjugates
US9732084B2 (en) 2010-04-15 2017-08-15 Medimmune Limited Pyrrolobenzodiazepines used to treat proliferative diseases
US10561739B2 (en) 2010-04-15 2020-02-18 Seattle Genetics Inc. Targeted pyrrolobenzodiazapine conjugates
US9399641B2 (en) 2011-09-20 2016-07-26 Medimmune Limited Pyrrolobenzodiazepines as unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates
US9388187B2 (en) 2011-10-14 2016-07-12 Medimmune Limited Pyrrolobenzodiazepines
US9399073B2 (en) 2011-10-14 2016-07-26 Seattle Genetics, Inc. Pyrrolobenzodiazepines
US10329352B2 (en) 2011-10-14 2019-06-25 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US10328084B2 (en) 2011-10-14 2019-06-25 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9387259B2 (en) 2011-10-14 2016-07-12 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9526798B2 (en) 2011-10-14 2016-12-27 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9713647B2 (en) 2011-10-14 2017-07-25 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9707301B2 (en) 2011-10-14 2017-07-18 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9856323B2 (en) 2012-04-26 2018-01-02 Bioatla, Llc Anti-CD22 antibodies
EP2841459A4 (fr) * 2012-04-26 2016-04-13 Bioatla Llc Anticorps anti-cd22
US10335497B2 (en) 2012-10-12 2019-07-02 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10646584B2 (en) 2012-10-12 2020-05-12 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9415117B2 (en) 2012-10-12 2016-08-16 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9889207B2 (en) 2012-10-12 2018-02-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10994023B2 (en) 2012-10-12 2021-05-04 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US12121590B2 (en) 2012-10-12 2024-10-22 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9745303B2 (en) 2012-10-12 2017-08-29 Medimmune Limited Synthesis and intermediates of pyrrolobenzodiazepine derivatives for conjugation
EP2906251B1 (fr) * 2012-10-12 2017-09-27 ADC Therapeutics SA Conjugués anticorps anti-cd22 - pyrrolobenzodiazépine
US11701430B2 (en) 2012-10-12 2023-07-18 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9943610B2 (en) 2012-12-21 2018-04-17 Bioalliance C.V. Hydrophilic self-immolative linkers and conjugates thereof
US9562049B2 (en) 2012-12-21 2017-02-07 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9408923B2 (en) 2012-12-21 2016-08-09 Bioalliance C.V. Hydrophilic self-immolative linkers and conjugates thereof
US9567340B2 (en) 2012-12-21 2017-02-14 Medimmune Limited Unsymmetrical pyrrolobenzodiazepines-dimers for use in the treatment of proliferative and autoimmune diseases
US10576164B2 (en) 2013-03-13 2020-03-03 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9821074B2 (en) 2013-03-13 2017-11-21 Genentech, Inc. Pyrrolobenzodiazepines and conjugates thereof
US9649390B2 (en) 2013-03-13 2017-05-16 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9956298B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9950077B2 (en) 2014-06-20 2018-04-24 Bioalliance C.V. Anti-folate receptor alpha (FRA) antibody-drug conjugates and methods of using thereof
CN106661123A (zh) * 2014-06-20 2017-05-10 荷商台医(有限合伙)公司 抗‑cd22抗体‑药物缀合物及其使用方法
WO2015196089A1 (fr) * 2014-06-20 2015-12-23 Bioalliance C.V. Conjugués anticorps anti-cd22-médicament et leurs méthodes d'utilisation
EP3157961A4 (fr) * 2014-06-20 2018-01-24 BioAlliance C.V. Conjugués anticorps anti-cd22-médicament et leurs méthodes d'utilisation
US10556966B2 (en) 2014-09-12 2020-02-11 Genentech, Inc. Anti-HER2 antibodies and immunoconjugates
US10179820B2 (en) 2014-09-12 2019-01-15 Genentech, Inc. Anti-HER2 antibodies and immunoconjugates
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
CN108093640A (zh) * 2015-04-15 2018-05-29 Adc治疗股份有限公司 位点特异性抗体-药物缀合物
WO2016166307A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués anticorps-médicaments spécifiques de sites
WO2016166304A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués anticorps-médicaments spécifiques de sites
CN108093640B (zh) * 2015-04-15 2022-05-17 Adc治疗股份有限公司 位点特异性抗体-药物缀合物
KR20170137116A (ko) * 2015-04-15 2017-12-12 에이디씨 테라퓨틱스 에스에이 부위-특이적 항체-약물 접합체
WO2016166301A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués de médicament-anticorps spécifiques de site
US11702473B2 (en) 2015-04-15 2023-07-18 Medimmune Limited Site-specific antibody-drug conjugates
KR102315256B1 (ko) 2015-04-15 2021-10-21 에이디씨 테라퓨틱스 에스에이 부위-특이적 항체-약물 접합체
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
AU2016247584B2 (en) * 2015-04-15 2021-01-28 Adc Therapeutics S.A. Site-specific antibody-drug conjugates
JP2018516860A (ja) * 2015-04-15 2018-06-28 メディミューン リミテッド 部位特異的な抗体−薬物複合体
US10058613B2 (en) 2015-10-02 2018-08-28 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
US10639373B2 (en) 2015-10-02 2020-05-05 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
US10632196B2 (en) 2015-10-02 2020-04-28 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
US10472422B2 (en) 2016-01-08 2019-11-12 Abgenomics International Inc. Tetravalent anti-PSGL-1 antibodies and uses thereof
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11384098B2 (en) 2017-02-08 2022-07-12 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11813335B2 (en) 2017-02-08 2023-11-14 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11596696B2 (en) 2017-04-20 2023-03-07 Adc Therapeutics Sa Combination therapy with an anti-CD25 antibody-drug conjugate
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11426467B2 (en) 2017-06-14 2022-08-30 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD25 ADC
US11938192B2 (en) 2017-06-14 2024-03-26 Medimmune Limited Dosage regimes for the administration of an anti-CD19 ADC
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11583590B2 (en) 2017-09-29 2023-02-21 Daiichi Sankyo Company, Limited Antibody-pyrrolobenzodiazepine derivative conjugate and method of use thereof for treating a tumor
US11628223B2 (en) 2017-09-29 2023-04-18 Daiichi Sankyo Company, Limited Antibody-drug conjugates comprising substituted benzo[e]pyrrolo[1,2-α][1,4]diazepines
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
US11484606B2 (en) 2019-06-07 2022-11-01 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11752197B2 (en) 2019-08-12 2023-09-12 Regeneron Pharmaceuticals, Inc. Macrophage stimulating 1 receptor (MST1R) variants and uses thereof
US11976122B2 (en) 2020-07-31 2024-05-07 Adc Therapeutics Sa Anti-IL13Rα2 antibodies

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