US20180086828A1 - Site-specific antibody-drug conjugates - Google Patents

Site-specific antibody-drug conjugates Download PDF

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US20180086828A1
US20180086828A1 US15/566,550 US201615566550A US2018086828A1 US 20180086828 A1 US20180086828 A1 US 20180086828A1 US 201615566550 A US201615566550 A US 201615566550A US 2018086828 A1 US2018086828 A1 US 2018086828A1
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amino acid
conjugate according
cysteine
antibody
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Patricius Hendrikus Cornelis Van Berkel
Philip Wilson Howard
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ADC Therapeutics SA
MedImmune Ltd
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ADC Therapeutics SA
MedImmune Ltd
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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
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • 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
    • 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
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present disclosure relates to site-specific antibody-drug conjugates.
  • Conjugates comprising pyrrolobenzodiazepines (PBDs) having a labile protecting group in the form of a linker to the antibody which binds CD22 are described.
  • PBDs pyrrolobenzodiazepines
  • ADC antibody-drug conjugates
  • cytotoxic or cytostatic agents i.e. drugs to kill or inhibit tumor cells in the treatment of cancer
  • targets delivery of the drug moiety to tumors, and intracellular accumulation therein Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No.
  • the present inventors have developed particular antibody-drug conjugates in which the antibody moiety is modified so as to increase the safety and efficacy of the ADC.
  • cytotoxic drugs have typically been conjugated to the antibodies in a non-site-specific manner via lysine side chains or by reducing interchain disulfide bonds present in the antibodies to provide activated native cysteine sulfhydryl groups.
  • Site-specific conjugation of drug to antibody has also been considered with a view to provide ADC populations with high homogeneity and batch-to-batch consistency with respect to drug-to-antibody ratio (DAR) and attachment site.
  • Site-specific attachment has typically been achieved by substituting a native amino acid in the antibody with an amino acid such as cysteine, to which a drug moiety can be conjugated (see Piel et al., JBC, Vol. 275, No. 39, Issue of September 29, pp. 30445-30450—conjugation of an IgG S442C variant with bromoacetyl-TMT); also Junutula et al., Nature Biotechnology, vol. 26, no. 8, pp. 925-932).
  • WO2013/093809 discusses a number of engineered antibody constant regions, a sub-set of which are exemplified as part of conjugates to cytotoxic drugs such as monomethyl auristatin D (MMAD).
  • MMAD monomethyl auristatin D
  • WO2011/005481 describes engineered antibody Fc regions for site-specific conjugation, including exemplification of biotin-PEG2-maleimide to a number of he engineered antibodies.
  • WO2006-065533 describes antibody Fc regions in which one or more of the ‘native’ interchain-disulphide-forming cysteines present in the heavy and/or light chain is substituted with another amino acid, so as to leave the complementary cysteine sulphydryl available for conjugation to a drug moiety.
  • the present inventors have developed particular antibody-drug conjugates in which the drug moiety is conjugated in a site-specific manner.
  • the present inventors have found that antibody-drug conjugates where the Drug unit (D L ) is conjugated to particular interchain cysteine residues have unexpected and advantageous properties.
  • these newly developed ADCs have advantageous manufacturing and pharmacological properties which are described herein.
  • the antibody of the conjugates described herein comprises one or more substitution of an interchain cysteine residue by an amino acid that is not cysteine.
  • the antibody of the conjugates described herein retains at least one unsubstituted interchain cysteine residue for conjugation of the drug moiety to the antibody.
  • the number of retained interchain cysteine residues in the antibody is greater than zero but less than the total number of interchain cysteine residues in the parent (native) antibody.
  • the antibody has at least one, at least two, at least three, at least four, at least five, at least six or at least seven interchain cysteine residues.
  • the antibody has an even integral number of interchain cysteine residues (e.g., at least two, four, six or eight). In some embodiments, the antibody has less than eight interchain cysteine residues.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by an amino acid that is not cysteine.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.130.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprise heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein (i) has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein (i) has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprises light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprises heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • antibody-drug conjugates wherein the antibody comprises specific mutations, or combinations of mutations, in the heavy chain have unexpected and advantageous properties.
  • the present inventors have identified antibody mutations in the heavy chain which reduce the toxicity and increase the serum half-lives of the ADCs they are incorporated into, as compared to otherwise identical ADCs comprising antibodies which lack the specific mutations.
  • the present inventors have identified the Leucine residues at positions 234 and 235 in the EU index set forth in Kabat (residues L117 and L118 in SEQ ID NO.110) as residues which, when substituted by an amino acid that is not leucine, allow for ADCs with advantageous properties.
  • the antibody of the conjugates described herein comprises a heavy chain having a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid (that is, an amino acid that is not identical to that found in the ‘wild-type’ sequence).
  • a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid (that is, an amino acid that is not identical to that found in the ‘wild-type’ sequence).
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted by any other amino acid.
  • the antibody is an IgG1 isotype and the leucine at position 234 in the EU index set forth in Kabat and/or the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine.
  • the leucines at position 234 and 235 in the EU index set forth in Kabat are substituted by an amino acid that is not leucine, such as alanine.
  • One or both Leucines may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, wherein the leucine at position 117 and/or the leucine at position 118 is substituted by an amino acid that is not leucine, such as alanine.
  • the leucine at position 117 and/or the leucine at position 118 is substituted by an amino acid that is not leucine, such as alanine.
  • both the leucines at position 117 and 118 are substituted by an amino acid that is not leucine, such as alanine.
  • One or both Leucines may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody is an IgG3 isotype and the leucine at position 234 in the EU index set forth in Kabat and/or the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine.
  • the leucines at position 234 and 235 in the EU index set forth in Kabat are substituted by an amino acid that is not leucine, such as alanine.
  • One or both Leucines may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, wherein the leucine at position 164 and/or the leucine at position 165 is substituted by an amino acid that is not leucine, such as alanine.
  • the leucines at position 164 and 165 are substituted by an amino acid that is not leucine, such as alanine.
  • One or both Leucines may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody is an IgG4 isotype and the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine, such as alanine.
  • the Leucine may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, wherein the leucine at position 115 is substituted by an amino acid that is not leucine, such as alanine.
  • the Leucine may be also substituted by other amino acids which are not Leucine, such as Glycine, Valine, or Isoleucine.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each retaining the unsubstituted interchain cysteine located in the C L domain, (iii) comprise heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprises light chains each retaining the unsubstituted interchain cysteine located in the C L domain, (iii) comprises heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprises heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • FIG. 1 Comparative systemic toxicitiy of site-specific ADCs, as described in Example 7.
  • conjugates comprising a pyrrolobenzodiazepine (PBD) drug moiety with a labile C2 or N10 protecting group and an antibody which binds CD22, wherein the antibody comprises an amino acid substitution of an interchain cysteine residue by an amino acid that is not cysteine, and wherein the drug moiety is conjugated to an interchain cysteine residue.
  • PBD pyrrolobenzodiazepine
  • conjugates comprising the antibodies described herein conjugated to other (i.e. non-PBD) functional moieties.
  • a functional moiety include a drug (PBD or non-PBD), a reporter, an organic moiety, and/or a binding moiety.
  • conjugates comprising an antibody fragment as described herein, along with pharmaceutical compositions comprising the conjugates.
  • Example antibodies or antibody fragment include scFv-Fc fusions and minibodies. Methods of preparing the conjugates and using the conjugates are disclosed, along with methods of using the conjugates to treat a number of diseases.
  • the conjugates described herein comprise a PBD drug moiety.
  • PBDs pyrrolobenzodiazepines
  • Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu.
  • PBDs are of the general structure:
  • 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.
  • WO 2011/130613 and WO 2011/130616 describe dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody.
  • 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.
  • conjugates where the Drug unit (D L ) is conjugated to particular interchain cysteine residues have unexpected and advantageous properties including increased efficacy and stability, improved ease of manufacture, and reduced systemic toxicity.
  • the disclosure provides a conjugate of formula L-(DL)p, where DL is of formula I or II::
  • L is an antibody (Ab) which binds CD22;
  • R 12 is selected from the group consisting of:
  • each of R 21 , R 22 and R 23 are independently selected from H, C 1-3 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;
  • 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; pyridyl; and thiophenyl; and
  • R 24 is selected from: H; C 1-3 saturated alkyl; C 2-3 alkenyl; C 1-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
  • R 12 is
  • R 26a and R 26b are independently selected from H, F, C 1-4 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 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo;
  • R and R′ are independently selected from optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups;
  • R 7 is selected from H, R, OH, OR, SH, SR, NH 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 C 1-4 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:
  • each of R 11 , R 12 and R 13 are independently selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 2 group is no more than 5;
  • 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 16a and R 16b are independently selected from H, F, C 1-4 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 16a and R 16b is H, the other is selected from nitrile and a C 1-4 alkyl ester;
  • R 22 is of formula IIIa, formula IIIb or formula IIIc:
  • A is a C 5-7 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 C 1-2 alkyl;
  • Q is selected from O—R L2′ , S—R L2′ and NR N —R L2′ , and R N is selected from H, methyl and ethyl
  • X is selected from the group comprising: O—R L2′ , S—R L2′ , CO 2 —R L2′ , CO—R L2′ , NH—C( ⁇ O)—R L2′ , NHNH—R L2′ , CONHNH—R L2′ ,
  • R N is selected from the group comprising H and C 1-4 alkyl
  • 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, ConjE, ConjF, ConjG and ConjH:
  • the link to the moiety shown is via a free S (active thiol) of an interchain cysteine residue on the cell binding agent.
  • the Conjugates comprise an antibody (Ab) as defined herein 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. Accordingly, also described herein are 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 disclosure provides a method of making a conjugate according to the first aspect of the disclosure comprising conjugating a compound of formula I L or II L :
  • R L1 is a linker suitable for conjugation to the antibody (Ab);
  • R 22L is of formula IIIa L , formula IIIb L or formula IIIc L :
  • Q L is selected from O—R L2 , S—R L2 and NR N —R L2 , and R N is sleected from H, methyl and ethyl
  • X L is selected from the group comprising: O—R L2 , S—R L2 , CO 2 —R L2 , and CO—R L2 , and N ⁇ C ⁇ O—R L2 , NHNH—R L2 , and CONHNH—R 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 disclosure provides a method of making a conjugate selected from the group consisting of ConjA, ConjB, ConjC, ConjD, ConjE, ConjF, ConjG and ConjH comprising conjugating a compound which is selected respectively from
  • WO 2011/130613 discloses compound 51:
  • WO 2013/041606 discloses Compound F (see compound 13e in WO 2013/041606).
  • Compound F 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 in compounds F and G is attached to the C2-phenyl group in the para rather than meta position.
  • Compound H has a cleavable protecting group on the second imine group which avoids cross-reactions during its synthesis and in the final product avoids the formation of carbinolamine and carbinolamine methyl ethers. This protection also avoids the presence of an reactive imine group in the molecule.
  • Compounds A, B, C, D, E, F, G and H 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 2011/130613, WO 2011/130598, WO 2013/041606 and WO 2011/130616 may be used in the present disclosure, and are incorporated herein by reference.
  • the drug linkers described herein may be synthesised as described in these disclosures.
  • the present disclosure is suitable for use in providing a PBD compound to a preferred site in a subject.
  • the conjugate may allow the release of an active PBD compound that does not retain any part of the linker. In such as case there is no stub present that could affect the reactivity of the PBD compound.
  • the speficied link between the PBD dimer and the antibody, in the present disclosure 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 specific 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 in vitro cytotoxicity, mass spectroscopy, HPLC, and the separation/analysis technique LC/MS.
  • Delivery of the compounds of formulae RelA, RelB, RelC, RelD, RelE or RelG is achieved at the desired activation site of the conjugates of formulae ConjA, ConjB, ConjC, ConjD, ConjE, ConhF, ConjG or ConjH 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 Substitution of Interchain Cysteine Residues
  • the antibody of the conjugates described herein comprise an amino acid substitution of an interchain cysteine residue by an amino acid that is not cysteine.
  • Naturally occurring antibodies generally include two larger heavy chains and two smaller light chains. In the case of native full-length antibodies, these chains join together to form a “Y-shaped” protein.
  • Heavy chains and light chains include cysteine amino acids that can be joined to one another via disulphide linkages. Heavy chains are joined to one another in an antibody by disulphide linkages between cysteine amino acids in each chain. Light chains are joined to heavy chains also by disulphide linkages between cysteine amino acids in the chains. Such disulphide linkages generally are formed between thiol side chain moieties of the free cysteine amino acids.
  • the cysteine amino acids which typically take part in these interchain disulphide linkages in naturally occurring antibodies are described herein as “interchain cysteine residues” or “interchain cysteines”.
  • cysteine amino acids in each IgG1 isotype heavy chain (‘HC’-220, 226, and 229 in the EU index set forth in Kabat) and one particular cysteine in each light chain (‘LC’- ⁇ (kappa)214 or ⁇ (lambda)213) are “interchain cysteines” as they generally participate in disulphide linkages between the antibody chains.
  • the interchain cysteine residues are located in the CL domain of the light chain, the CH 1 domain of the heavy chain, and in the hinge region.
  • the number of interchain cysteine residues in an antibody depends on the antibody isotype.
  • the antibody of the conjugates described herein comprise an amino acid substitution of an interchain cysteine residue by an amino acid that is not cysteine.
  • the amino acid substituted for an interchain cysteine typically does not include a thiol moiety, and often is a valine, serine, threonine, alanine, glycine, leucine, isoleucine, other naturally occurring amino acid, or non-naturally occurring amino acid.
  • the amino acid substitution is a valine for the interchain cysteine residue.
  • one or more or all interchain cysteines are ‘substituted’ for no amino acid; that is, the one or more or all interchain cysteines is deleted and not replaced by another amino acid.
  • SEQ ID NO.153 as disclosed herein is an example of “a light chain comprising the amino acid sequence of SEQ ID NO. 150 wherein the cysteine at position 105 in SEQ ID NO: 150, is substituted by an amino acid that is not cysteine” wherein the cysteine is substituted for no amino acid i.e. deleted.
  • the serine at positon 103 is also preferably deleted. See, for example, SEQ ID NO: 163.
  • substituted and a substitution as used herein in reference to amino acids is used to mean the replacement of an amino acid residue with a different—that is, non-identical—amino acid residue (or with no amino acid residue—that is, a deletion—as explained above).
  • substitution of a leucine by an amino acid which is not leucine means the replacement of the specified with any non-leucine amino acid.
  • This can be—for example—Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, Cys, Gly, Ala, Val, Ile, Phe, Trp, Pro, or Met, but is preferably Gly, Ala, Val, or Ile, and most preferably Ala,
  • the antibody of the conjugates described herein retains at least one unsubstituted interchain cysteine residue for conjugation of the drug moiety to the antibody.
  • the number of retained interchain cysteine residues in the antibody is greater than zero but less than the total number of interchain cysteine residues in the parent (native) antibody.
  • the antibody has at least one, at least two, at least three, at least four, at least five, at least six or at least seven interchain cysteine residues.
  • the antibody has an even integral number of interchain cysteine residues (e.g., at least two, four, six or eight). In some embodiments, the antibody has less than eight interchain cysteine residues.
  • the antibody of the conjugates described herein retains the unsubstituted hinge region interchain cysteines.
  • the antibody retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines.
  • the antibody has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein retains at least one unsubstituted hinge region interchain cysteine.
  • the antibody retains an unsubstituted HC226 according to the EU index as set forth in Kabat.
  • the antibody retains an unsubstituted HC229 according to the EU index as set forth in Kabat.
  • each heavy chain retains exactly one (i.e. not more than one) unsubstituted hinge region interchain cysteine.
  • the antibody of the conjugates described herein has the amino acid substitution of valine for each of the hinge region interchain cysteines.
  • the antibody has the amino acid substitution of valine each of HC226 and HC229 according to the EU index as set forth in Kabat
  • the antibody of the conjugates described herein comprise: (i) a light chain having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (ii) a heavy chain retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein comprise: (i) a light chain having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (ii) a heavy chain retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprise: (i) light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (ii) heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein comprise: (i) light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (ii) heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprise: (i) a light chain retaining the unsubstituted interchain cysteine located in the C L domain, and (ii) a heavy chain having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein comprise: (i) a light chain retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (ii) a heavy chain having an amino acid substitution of the interchain cysteine residue HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprise: (i) light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (ii) heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein comprise: (i) light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (ii) heavy chains each having an amino acid substitution of the interchain cysteine residue HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise a light chain having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise a heavy chain retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise a light chain having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise a heavy chain retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise a light chain retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprise a heavy chain having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise a light chain retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise a heavy chain having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprise heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprise a light chain having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise a heavy chain retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise a light chain having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise a heavy chain retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has the amino acid substitution of valine for each of the hinge region interchain cysteines, (ii) comprises a light chain having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprises a heavy chain retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein has the amino acid substitution of valine for each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises a light chain having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises a heavy chain retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has the amino acid substitution of valine for each of the hinge region interchain cysteines, (ii) comprises light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, and (iii) comprises heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain.
  • the antibody of the conjugates described herein has the amino acid substitution of valine for each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has the amino acid substitution of valine for each of the hinge region interchain cysteines, (ii) comprises a light chain retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprises a heavy chain having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises a light chain retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises a heavy chain having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprises light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprises heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprises a light chain retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprises a heavy chain having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein has the amino acid substitution of valine for each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises a light chain retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises a heavy chain having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) has the amino acid substitution of valine for each of the hinge region interchain cysteines, (ii) comprises light chains each retaining the unsubstituted interchain cysteine located in the C L domain, and (iii) comprises heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain.
  • the antibody of the conjugates described herein has the amino acid substitution of valine for each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, and (iii) comprises heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • Table 1 illustrates positions of interchain cysteines in the heavy chain constant region and light chain constant region of particular antibody isotypes according to the EU index as set forth in Kabat and with reference to the sequences disclosed herein.
  • Each of the interchain cysteine positions present in an antibody or antibody fragment may be substituted with an amino acid that is not a cysteine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110 or fragment thereof, SEQ ID NO.120 or fragment thereof, SEQ ID NO.130 or fragment thereof, or SEQ ID NO.140 or fragment thereof.
  • the drug moiety is conjugated to the cysteine at position 103 of SEQ ID NO.110, the cysteine at position 14 of SEQ ID NO.120, the cysteine at position 14 of SEQ ID NO.130, or the cysteine at position 14 of SEQ ID NO.140.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, or fragment thereof, wherein the cysteine at position 103 of SEQ ID NO.110, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO. 111 discloses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein the cysteine at position 103 of SEQ ID NO.110 is substituted by a serine residue.
  • SEQ ID NO. 112 discloses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein the cysteine at position 103 of SEQ ID NO.110 is substituted by a valine residue.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, or fragment thereof, wherein the cysteine at positions 14 of SEQ ID NO.120, if present, is substituted by an amino acid that is not cysteine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, or fragment thereof, wherein the cysteine at position 14 in SEQ ID NO: 130, if present, is substituted by an amino acid that is not cysteine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, or fragment thereof, wherein the cysteine at position 14 in SEQ ID NO: 140, if present, is substituted by an amino acid that is not cysteine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, or fragment thereof, wherein each of the cysteines at positions 109 and 112 in SEQ ID NO: 110, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO: 113 dislcoses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is substituted by a serine residue.
  • SEQ ID NO: 114 dislcoses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein each of the cysteines at positions 109 and 112 in SEQ ID NO: 110 is substituted by a valine residue.
  • the drug moiety is conjugated to the cysteine at position 103 of SEQ ID NO.110.
  • the cysteine at position 109 in SEQ ID NO: 110, if present, is substituted by an amino acid that is not cysteine, and the cysteine at position 112 in SEQ ID NO: 110, if present, is unsubstituted.
  • the cysteine at position 112 in SEQ ID NO: 110 is substituted by an amino acid that is not cysteine, and the cysteine at position 109 in SEQ ID NO: 110, if present, is unsubstituted.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, or fragment thereof, wherein each of the cysteines at positions 103, 106, and 109 in SEQ ID NO: 120, if present, is substituted by an amino acid that is not cysteine.
  • the cysteine at position 102 in SEQ ID NO: 120, if present, is also substituted by an amino acid that is not cysteine.
  • all but one of the cysteines at positions 103, 106, 109, and 102 in SEQ ID NO: 120, if present, are substituted by an amino acid that is not cysteine.
  • the cysteine at position 103, 106, 109, or 102 in SEQ ID NO: 120, if present, is unsubstituted.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.120.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, or fragment thereof, wherein each of the cysteines at positions 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, is substituted by an amino acid that is not cysteine. In some embodiments, all but one of the cysteines at positions 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, are substituted by an amino acid that is not cysteine.
  • the cysteine at position 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, or 159 in SEQ ID NO: 130, if present, is unsubstituted.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.130.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, or fragment thereof, wherein each of the cysteines at positions 106 and 109 in SEQ ID NO: 140, if present, is substituted by an amino acid that is not cysteine.
  • the cysteine at position 106 in SEQ ID NO: 140, if present, is substituted by an amino acid that is not cysteine, and the cysteine at position 109 in SEQ ID NO: 140, if present, is unsubstituted.
  • the cysteine at position 109 in SEQ ID NO: 140 is substituted by an amino acid that is not cysteine, and the cysteine at position 106 in SEQ ID NO: 140, if present, is unsubstituted.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.140.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, or fragment thereof, wherein each of the cysteines at positions 103, 109 and 112 in SEQ ID NO: 110, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO: 115 discloses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein each of the cysteines at positions 103, 109 and 112 in SEQ ID NO: 110 is substituted by a serine residue.
  • SEQ ID NO: 116 discloses a heavy chain comprising the amino acid sequence of SEQ ID NO.110 wherein each of the cysteines at positions 103, 109 and 112 in SEQ ID NO: 110 is substituted by a valine residue.
  • the cysteine at position 109 in SEQ ID NO: 110 if present, is substituted by an amino acid that is not cysteine, and the cysteine at position 112 in SEQ ID NO: 110, if present, is unsubstituted.
  • the cysteine at position 112 in SEQ ID NO: 110 is substituted by an amino acid that is not cysteine, and the cysteine at position 109 in SEQ ID NO: 110, if present, is unsubstituted.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, or fragment thereof, wherein each of the cysteines at positions 14, 103, 106 and 109 in SEQ ID NO: 120, if present, is substituted by an amino acid that is not cysteine.
  • all but one of the cysteines at positions 103, 106, 109, and 102 in SEQ ID NO: 120, if present, are substituted by an amino acid that is not cysteine.
  • the cysteine at position 103, 106, 109, or 102 in SEQ ID NO: 120, if present is unsubstituted.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, or fragment thereof, wherein each of the cysteines at positions 14, 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, is substituted by an amino acid that is not cysteine. In some embodiments, all but one of the cysteines at positions 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, and 159 in SEQ ID NO: 130, if present, are substituted by an amino acid that is not cysteine.
  • the cysteine at position 111, 114, 120, 126, 129, 135, 141, 144, 150, 156, or 159 in SEQ ID NO: 130, if present, is unsubstituted.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, or fragment thereof, wherein each of the cysteines at positions 14, 106, and 109 in SEQ ID NO: 140, if present, is substituted by an amino acid that is not cysteine.
  • the cysteine at position 106 in SEQ ID NO: 140, if present, is substituted by an amino acid that is not cysteine, and the cysteine at position 109 in SEQ ID NO: 140, if present, is unsubstituted.
  • the cysteine at position 109 in SEQ ID NO: 140 is substituted by an amino acid that is not cysteine, and the cysteine at position 106 in SEQ ID NO: 140, if present, is unsubstituted.
  • the antibody of the conjugates described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO. 150, or fragment thereof, or SEQ ID NO. 160 or fragment thereof.
  • the drug moiety is conjugated to the cysteine at position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
  • the antibody of the conjugates described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO. 150, or fragment thereof, wherein the cysteine at position 105, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO. 151 discloses a light chain comprising the amino acid sequence of SEQ ID NO. 150 wherein the cysteine at position 105 is substituted by a serine residue.
  • SEQ ID NO. 152 discloses a light chain comprising the amino acid sequence of SEQ ID NO. 150 wherein the cysteine at position 105 is substituted by a valine residue.
  • SEQ ID NO. 153 discloses a light chain having the amino acid sequence of SEQ ID NO. 150, wherein the cysteine at position 105 has been deleted.
  • the antibody of the conjugates described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO. 160, or fragment thereof, wherein the cysteine at position 102, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO. 161 discloses a light chain comprising the amino acid sequence of SEQ ID NO. 160 wherein the cysteine at position 102 is substituted by a serine residue.
  • SEQ ID NO. 162 discloses a light chain comprising the amino acid sequence of SEQ ID NO. 160 wherein the cysteine at position 102 is substituted by a valine residue.
  • SEQ ID NO. 163 discloses a light chain having the amino acid sequence of SEQ ID NO. 160, wherein the cysteine at position 102 and the serine at position 103 have been deleted.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 103 of SEQ ID NO.110.
  • cysteines at positions 109 and 112 in SEQ ID NO: 110 are substituted for valine.
  • cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by serine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the cysteine at position 102 in SEQ ID NO: 120 is also substituted by an amino acid that is not cysteine.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.120.
  • cysteines at positions 103, 106, and 109 in SEQ ID NO: 120 are substituted for valine.
  • the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by serine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the cysteine at position 102 in SEQ ID NO: 120 is also substituted by an amino acid that is not cysteine.
  • the drug moiety is conjugated to the cysteine at position 103 of SEQ ID NO.120.
  • cysteines at positions 14, 106, and 109 in SEQ ID NO: 120 are substituted for valine.
  • cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by serine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.130.
  • cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by serine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.140.
  • the drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.140.
  • each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 are substituted for valine.
  • the cysteine at position 105 in SEQ ID NO: 150 or the cysteine at position 102 in SEQ ID NO: 160 is substituted by serine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the drug moiety is conjugated to the cysteine at position 105 of SEQ ID NO.150, the cysteine at position 102 of SEQ ID NO.160.
  • the Antibody Substitution of Kabat EU Residues 234 and/or 235
  • the antibody of the conjugates described herein comprises a heavy chain having a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat. It has been unexpectedly found that ADCs in which the antibody bears one, or preferably both, of these substitutions have improved tolerability and increased serum half-lives as compared to otherwise identical ADCs comprising antibodies which lack the specific mutations.
  • the ADCs disclosed herein which comprise a heavy chain having substitutions of the residues at positions 234 and 235 in the EU index set forth in Kabat actually have increased serum half-lives as compared to otherwise identical ADCs comprising antibodies which lack the mutations.
  • the ADCs comprising a heavy chain having substitutions of the residues at positions 234 and 235 in the EU index set forth also exhibit improved tolerability / reduced toxicity as compared to otherwise identical ADCs comprising antibodies which lack the mutations.
  • the antibody of the conjugates described herein comprises a heavy chain having a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted by any other amino acid.
  • the antibody is an IgG1 isotype and the leucine at position 234 in the EU index set forth in Kabat and/or the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine.
  • the antibody is an IgG3 isotype and the leucine at position 234 in the EU index set forth in Kabat and/or the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine.
  • the antibody is an IgG4 isotype and the leucine at position 235 in the EU index set forth in Kabat is substituted by an amino acid that is not leucine, such as alanine.
  • Table 2 illustrates positions of corresponding residues in the heavy chain constant region of particular antibody isotypes according to the EU index as set forth in Kabat and with reference to the sequences disclosed herein.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, wherein the leucine at position 117 and/or the leucine at position 118 is substituted by an amino acid that is not leucine, such as alanine.
  • the leucines at position 117 and 118 are substituted by an amino acid that is not leucine, such as alanine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, wherein the leucine at position 164 and/or the leucine at position 165 is substituted by an amino acid that is not leucine, such as alanine.
  • the leucines at position 164 and 165 are substituted by an amino acid that is not leucine, such as alanine.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, wherein the leucine at position 115 is substituted by an amino acid that is not leucine, such as alanine.
  • the Antibody Substitution of Interchain Cysteine Residues Combined with Substitution of Kabat EU Residues 234 and/or 235
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein (i) retain the unsubstituted hinge region interchain cysteines, (ii) comprise light chains each retaining the unsubstituted interchain cysteine located in the C L domain, (iii) comprise heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein retains unsubstituted HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue located in the C L domain, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprise light chains each having an amino acid substitution of the interchain cysteine residue ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprise heavy chains each retaining the unsubstituted interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the CH 1 domain, for example to HC220 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of the hinge region interchain cysteines, (ii) comprises light chains each retaining the unsubstituted interchain cysteine located in the C L domain, (iii) comprises heavy chains each having an amino acid substitution of the interchain cysteine residue located in the CH 1 domain, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat.
  • the antibody of the conjugates described herein has an amino acid substitution of each of HC226 and HC229 according to the EU index as set forth in Kabat, (ii) comprises light chains each retaining the unsubstituted interchain cysteine ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat, (iii) comprises heavy chains each having an amino acid substitution of interchain cysteine HC220 according to the EU index as set forth in Kabat, and (iv) comprise heavy chains each having an amino acid substitution of the the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat by any other amino acid.
  • both the residues at position 234 and 235 in the EU index set forth in Kabat are substituted.
  • the drug moiety is conjugated to the unsubstituted interchain cysteine located in the C L domain, for example to ⁇ LC214 or ⁇ LC213 according to the EU index as set forth in Kabat.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.110, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.140, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • the conjugates described herein have been found to be well-tolerated in in vivo disease models, allowing for reduced side-effects in subjects receiving the conjugates. Accordingly, in some embodiments the conjugates described herein have a higher MTD than an otherwise identical conjugate where the drug moieties are to the antibody at non-site specifically. MTD is typically tested in animals such as mouse (for example, Mus musculus ), rat (for example, Rattus norvegicus ), or monkey (for example, Macaca fascicularis ).
  • the conjugates described herein have an MTD in rat of at least 1 mg/kg delivered as a single-dose, for example at least 1.2 mg/kg, at least 1.4 mg/kg, at least 1.6 mg/kg, at least 1.8 mg/kg, at least 2.0 mg/kg, at least 2.2 mg/kg, at least 2.4 mg/kg, at least 2.6 mg/kg, at least 2.8 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, or at least 5.0 mg/kg delivered as a single-dose.
  • the site-specific conjugates described herein have an improved therapeutic index as compared to an otherwise identical non site-specific conjugate.
  • the therapeutic index for a site specific conjugate descried herein is at least 2% higher than an otherwise identical non site-specific conjugate. That is, if the non site-specific conjugate has a therapeutic index of 100:1, the site specific conjugate has a therapeutic index of at least 102:1.
  • the therapeutic index for a site specific conjugate descried herein is at least 5% higher than an otherwise identical non site-specific conjugate, for example at least 5% higher, at least 7% higher, at least 10% higher, at least 12% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 70% higher, at least 100% higher, at least 150% higher, or at least 200% higher than an otherwise identical non site-specific conjugate.
  • the relative systemic toxicity of a site-specific ADC newly described herein was compared to that of a known type of site-specific ADC—see Example 7 and FIG. 1 .
  • the site-specific ADC newly described herein was not observed to induce significant systemic toxicity, in contrast to the known site-specific ADC.
  • the site-specific conjugate has the same affinity for the cognate antigen as compared to an otherwise identical non site-specific conjugate. In some embodiments, the site-specific conjugate has a greater affinity for the cognate antigen as compared to an otherwise identical non site-specific conjugate.
  • the site-specific conjugate binds the cognate antigen with a dissociation constant (Kd) of at least 10 —6 M, such as at least 5 ⁇ 10 ⁇ 7 M, at least 10 ⁇ 7 M, at least 5 ⁇ 10 ⁇ 8 M, at least 10 ⁇ 9 M, such as at least 5 ⁇ 10 ⁇ 10 M, at least 10 ⁇ 10 M, at least 5 ⁇ 10 ⁇ 11 M, at least 10 ⁇ 11 M, at least 5 ⁇ 10 ⁇ 12 M, at least 10 ⁇ 12 M, at least 5 ⁇ 10 ⁇ 13 M, at least 10 ⁇ 13 M, at least 5 ⁇ 10 ⁇ 14 M, at least 10 ⁇ 14 M, at least 5 ⁇ 10 ⁇ 15 M, or at least 10 ⁇ 15 M.
  • Kd dissociation constant
  • the site-specific conjugate competitively inhibits the in vivo and/or in vitro binding to the cognate antigen of an otherwise identical non site-specific conjugate.
  • binds [antigen X] is used to mean the antibody binds [antigen X] with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 GI:3336842, record update date: Jan. 7, 2011 02:30 PM).
  • BSA Bovine Serum Albumin
  • the antibody binds [antigen X] with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 4 , 10 5 or 10 6 -fold higher than the antibody's association constant for BSA, when measured at physiological conditions.
  • the antibodies of the disclosure can typically bind [antigen X] with a high affinity.
  • the antibody can bind [antigen X] with a KD equal to or less than about 10 ⁇ 6 M, such as 1 ⁇ 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 , 10 ⁇ 10 , 10 ⁇ 11 , 10 ⁇ 12 , 10 ⁇ 13 or 10 ⁇ 14 M.
  • the site-specific conjugate has an EC50 of less than 35 ng/ml, such as less than 30 ng/ml, less than 25 ng/ml, less than 20 ng/ml, or less than 15 ng/ml. In some embodiments the EC50 of the site-specific conjugate is no higher than an otherwise identical non site-specific conjugate. In some embodiments the EC50 of the site-specific conjugate is at least 2 ng/ml lower than an otherwise identical non site-specific conjugate, for example at least 5 ng/ml lower, at least 10 ng/ml lower, at least 15 ng/ml lower, at least 20 ng/ml lower, at least 25 ng/ml lower, or at least 30 ng/ml lower.
  • Embodiments of the site-specific ADCs newly described herein allow for simplification of the ADC manufacture procedure.
  • cysteine engineered IgG version such as those described in Junutula et al., Nature Biotechnology, vol. 26, no. 8, pp. 925-932
  • additional cysteines are engineered into the IgG1 to allow for site-specific conjugation on the engineered cysteines.
  • the engineered cysteines are typically capped with other sulphydryl containing molecules such as GSH, cysteine etc.
  • the molecule In order to release the engineered cysteines for conjugation, the molecule must be reduced. This typically will also reduce the interchain disulphide bond between the heavy and light chains, as well as those in the hinge region.
  • the present disclosure specifically contemplates embodiemnts where the antibody comprises only two interchain cycteins suitable for conjugation (for example, one on each heavy chain) with the other interchain cycteine residues present in a native antibody having been substituted for an amino acid which is not cysteine.
  • This format allows the complex-reduction-reoxidation procedure described above to be dispensed with. Instead a straight forward reduction-conjugation procedure can be followed. This is possible because the site-specific antibody formats described herein typically do not contain interchain cysteines that are not ultimately intended to be conjugated to drug moiteies.
  • the site-specific antibody contains only two interchain cycteins suitable for conjugation (for example, one on each heavy chain). It is therefore not necessary to reoxidize the antibody molecule after the initial reduction step. Instead the molecule is reduced with a reducatant such as TCEP which reduces the (two) remaining interchain cysteines (with the other interchain cysteines having been substituted for amino acids which are not cysteine). The reduced cysteine sulphhydryl moiteis can then be conjugated to the drug-linker.
  • a reducatant such as TCEP which reduces the (two) remaining interchain cysteines (with the other interchain cysteines having been substituted for amino acids which are not cysteine).
  • the reduced cysteine sulphhydryl moiteis can then be conjugated to the drug-linker.
  • the newly described site-specific ADCs also avoid other potential manufacturing problems. For example, during the analysis of cysteine engineered IgGs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, the existence of Triple Light Chain antibodies (3LC) has been observed; the 3LC species appears to be the product of a disulfide bond formed between an extra light chain and an additional cysteine engineered into an IgG (Gomez et al., Biotechnol. Bioeng. 105(4)_748-60 (2010); Gomez et al., Biotechnol. Prog. 26(5)_1438-1445 (2010)). The newly described site-specific ADCs do not have inseted cysteines in the light chain, so have no potential to form contamination 3LC species.
  • conjugates in which the antibody comprises a heavy chain having a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat have improved terminal half-life as compared to another otherwise identical conjugate lacking the 234/235 substitution(s).
  • the terminal-half life may be measured as described herein in Example 6.
  • conjugates in which the antibody comprises a heavy chain having a substitution of the residue at position 234 in the EU index set forth in Kabat and/or a substitution of the residue at position 235 in the EU index set forth in Kabat have a half-life which is at least 110% of the half-life of an otherwise identical conjugate lacking the 234/235 substitution(s); for example at least 115% of the half-life, at least 120% of the half-life, at least 125% of the half-life, at least 130% of the half-life, at least 135% of the half-life, at least 140% of the half-life, at least 145% of the half-life, at least 150% of the half-life, at least 160% of the half-life, at least 170% of the half-life, at least 180% of the half-life, at least 190% of the half-life, or at least 200% of the half-life of an otherwise identical conjugate lacking the 234/235 substitution(s).
  • the antibody of the conjugates described herein is an antibody (Ab) which binds CD22. That is, the conjugates described herein are conjugates comprising antibodies which specifically bind to CD22.
  • CD22 polypeptide corresponds to Genbank accession no. BAB15489, version no. BAB15489.1 GI:10439338, record update date: Sep. 11, 2006 11:24 PM.
  • the nucleic acid encoding CD22 polypeptide corresponds to Genbank accession no AK026467, version no. AK026467.1 GI:10439337, record update date: Sep. 11, 2006 11:24 PM.
  • the antibody is an antibody that binds to CD22, the antibody comprising a VH domain having the sequence according to SEQ ID NO. 1.
  • the antibody may further comprise a VL domain.
  • the antibody further comprises a VL domain having the sequence according to SEQ ID NO. 2.
  • the antibody comprises a VH domain paired with a VL domain, the VH and VL domains having the sequences of SEQ ID NO. 1 paired with SEQ ID NO. 2.
  • VH and VL domain(s) may pair so as to form an antibody antigen binding site that binds CD22.
  • the antibody is an intact antibody comprising a VH domain paired with a VL domain, the VH and VL domains having sequences of SEQ ID NO. 1 paired with SEQ ID NO. 2.
  • the antibody is an antibody as described herein which has been modified (or further modified) as described below.
  • the antibody is a humanised, deimmunised or resurfaced version of an antibody disclosed herein.
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.130, a light chain, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.140, a light chain, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • An antibody of the conjugates described herein comprising a heavy chain comprising the amino acid sequence of SEQ ID NO.110, a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160, a VH domain having the sequence SEQ ID NO. 1, and a VL domain having the sequence SEQ ID NO. 2;
  • the numbering of the amino acids used herein is according to the numbering system of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va., hereinafter “Kabat”).
  • the “EU index as set forth in Kabat” refers to the residue numbering of the human IgG 1 EU antibody as described in Kabat et al. supra.
  • sequence alignment programs such as NCBI BLAST® (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to align the sequences with IgG1 to determine which residues of the desired isoform correspond to the Kabat positions described herein.
  • antibody encompasses any molecule comprising an antibody antigen-binding site (as, for example, formed by a paired VH domain and a VL domain).
  • antibody encompasses monoclonal antibodies (including intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain antibodies (such as scFv fusions with CH3), antibody fragments that exhibit the desired biological activity (e.g.
  • Antibodies may be murine, human, humanized, chimeric, or derived from other species.
  • the antibody is a single-chain Fv antibody fused to a CH3 domain (scFv-CH3).
  • the antibody is a single-chain Fv antibody fused to a Fc region (scFv-Fc).
  • the antibody is a minibody.
  • An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • An antibody includes an intact immunoglobulin molecule or an immunologically active portion of a intact immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen binding site.
  • the antibody can be of any isotype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass, or allotype (e.g.
  • human G1m1, G1m2, G1m3, non-G1m1 [that, is any allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3) of antibody molecule.
  • the immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
  • an “intact antibody” herein is one comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.
  • antibody heavy chain constant region refers to the portion of an antibody molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
  • Fc region As used herein, the terms “Fc region”, “Fc domain” and “Fc” relate to the constant region of an antibody excluding the first constant region immunoglobulin domain and further relates to portions of that region. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains, or portions thereof. For IgA and IgM, Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 (C gamma 2 and C gamma 3) and the hinge between Cy1 (C gamma 1) and Cy2 (C gamma 2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, as numbered according to the numbering system of the EU index as set forth in Kabat et al. supra.
  • the Fc domain comprises from about amino acid residue 236 to about 447 of the human IgG1 constant domain.
  • Fc polypeptide may refer to this region in isolation, or this region in the context of an antibody, or an antigen-binding portion thereof, or Fc fusion protein.
  • the “intact heavy chain constant region” comprises the Fc region and further comprises the CH1 domain and hinge as well as the CH2 and CH3 (and, optionally, CH4 of IgA and IgE) domains of the IgG heavy chain.
  • “Hinge region” as used herein, is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, 1985, Malec. Immunol. 22: 161-206), and refers to the portion of an IgG molecule comprising the C-terminal portion of the CH1 domain and the N-terminal portion of the CH2 domain.
  • Exemplary hinge regions for human IgG1, IgG2, IgG2 and IgG4 and mouse IgG1 and IgG2A are provided in U.S. Pat. No. 6,165,476, at the Table shown at column 4, line 54 to column 5, line 15, and also illustrated, for example, in Janeway et al., 1999, Immunology: The Immune System in Health and Disease, 4th ed.
  • Hinge regions of other IgG isotypes may be aligned with the IgG 1 sequence by placing the first and last cysteine residues forming inter-heavy chain S—S bonds in the same positions.
  • the “lower hinge region” of an Fc region is normally defined as the stretch of residues immediately C-terminal to the hinge region, i.e. residues 233 to 239 of the Fe region.
  • IgG hinge-Fc region or “hinge-Fc fragment” as used herein refers to a hinge region (approximately residues 216-230) and an Fc region (residues 231-44 7) C-terminal thereto.
  • fragment is used herein to describe a portion of sequence that is shorter than the full-length sequence disclosed herein.
  • antibodies comprising “fragments” as disclosed herein retain the ability to bind the target antigen, most preferably with a specific binding activity of about 70% or more compared to of an otherwise identical antibody comprising the full-length sequence disclosed herein (for example, about 10% or more, 50% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the binding activity).
  • the specific binding activity is in vitro.
  • the specific binding activity sometimes is quantified by an in vitro homogeneous assay or an in vitro heterogeneous assay.
  • the specific binding activity is in vivo, and sometimes, the specific binding activity is determined in situ.
  • a “fragment” is at least 50 amino acids long, such as at least 75, at least 100, at least 150, at least 200, at least 250, or at least 300 amino acids long.
  • sequences of the antibody heavy chain variable regions and/or the light chain variable regions disclosed herein may be modified by substitution, insertion or deletion.
  • 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.
  • Preferred conservative substitutions are those wherein one amino acid is substituted for another within the groups of amino acids indicated herein below:
  • Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys)
  • Amino acids having non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met)
  • Amino acids having aliphatic side chains (Gly, Ala Val, Leu, Ile)
  • Amino acids having acidic side chains (Asp, Glu)
  • Particular preferred conservative amino acids substitution groups are: Val-Leu-Ile, Phe-Tyr, Lys-Arg, Ala-Val, and Asn-Gln.
  • the antibody of the conjugates described herein comprises a heavy chain having an amino acid sequence with 80% or more amino acid sequence identity (for example, about 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity) to a heavy chain described herein.
  • amino acid sequence identity for example, about 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity
  • the antibody of the conjugates described herein comprises a light chain having an amino acid sequence with 80% or more amino acid sequence identity (for example, about 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity) to a light chain described herein.
  • amino acid sequence identity for example, about 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity
  • the antibody of the conjugates described herein comprises a heavy chain having an amino acid sequence identical to the amino acid sequence of a heavy chain described herein, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., substitutions, insertions and/or deletions) relative to the amino acid sequence of the heavy chain described herein.
  • the antibody of the conjugates described herein comprises a light chain having an amino acid sequence identical to the amino acid sequence of a light chain described herein, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., substitutions, insertions and/or deletions) relative to the amino acid sequence of the light chain described herein.
  • the antibodies disclosed herein may be modified. For example, to make them less immunogenic to a human subject. This may be achieved using any of a number of techniques familiar to the person skilled in the art. Some of these techniques are described in more detail below.
  • a “humanized antibody” refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody wherein a portion of the variable region, preferably a portion substantially less than the intact human variable domain, has been substituted by the corresponding sequence from a non-human species and wherein the modified variable region is linked to at least another part of another protein, preferably the constant region of a human antibody.
  • the expression “humanized antibodies” includes human antibodies in which one or more complementarity determining region (“CDR”) amino acid residues and/or one or more framework region (“FW” or “FR”) amino acid residues are substituted by amino acid residues from analogous sites in rodent or other non-human antibodies.
  • the expression “humanized antibody” also includes an immunoglobulin amino acid sequence variant or fragment thereof that comprises an FR having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Or, looked at another way, a humanized antibody is a human antibody that also contains selected sequences from non-human (e.g. murine) antibodies in place of the human sequences.
  • a humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity.
  • Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
  • CDR grafting There are a range of humanisation techniques, including ‘CDR grafting’, ‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as ‘veneering’), ‘composite antibodies’, ‘Human String Content Optimisation’ and framework shuffling.
  • the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are ‘grafted’ onto the human framework).
  • CDR complementary-determining region
  • donor antibody such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties
  • donor antibody such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are ‘grafted’ onto the human framework).
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues (this may happen when, for example, a particular FR residue has significant effect on antigen binding).
  • humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin.
  • the method consists of combining the V H or V L domain of a given non-human antibody specific for a particular epitope with a human V H or V L library and specific human V domains are selected against the antigen of interest. This selected human VH is then combined with a VL library to generate a completely human VH ⁇ VL combination.
  • the method is described in Nature Biotechnology (N.Y.) 12, (1994) 899-903.
  • two or more segments of amino acid sequence from a human antibody are combined within the final antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations which limit or avoid human T cell epitopes in the final composite antibody V regions. Where required, T cell epitopes are limited or avoided by, exchanging V region segments contributing to or encoding a T cell epitope with alternative segments which avoid T cell epitopes. This method is described in US 2008/0206239 A1.
  • This method involves the removal of human (or other second species) T-cell epitopes from the V regions of the therapeutic antibody (or other molecule).
  • the therapeutic antibodies V-region sequence is analysed for the presence of MHC class II- binding motifs by, for example, comparison with databases of MHC-binding motifs (such as the “motifs” database hosted at www.wehi.edu.au).
  • MHC class II- binding motifs may be identified using computational threading methods such as those devised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutive overlapping peptides from the V-region sequences are testing for their binding energies to MHC class II proteins.
  • This data can then be combined with information on other sequence features which relate to successfully presented peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.
  • T-cell epitopes Once potential second species (e.g. human) T-cell epitopes have been identified, they are eliminated by the alteration of one or more amino acids.
  • the modified amino acids are usually within the T-cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and therefore, may not be adjacent in the primary structure). Most typically, the alteration is by way of substitution but, in some circumstances amino acid addition or deletion will be more appropriate.
  • This method involves:
  • the method compares the non-human sequence with the functional human germline gene repertoire. Those human genes encoding canonical structures identical or closely related to the non-human sequences are selected. Those selected human genes with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these human FRs. This method is described in patent WO 2005/079479 A2.
  • This method compares the non-human (e.g. mouse) sequence with the repertoire of human germline genes and the differences are scored as Human String Content (HSC) that quantifies a sequence at the level of potential MHC/T-cell epitopes.
  • HSC Human String Content
  • the target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).
  • the CDRs of the non-human antibody are fused in-frame to cDNA pools encompassing all known heavy and light chain human germline gene frameworks. Humanised antibodies are then selected by e.g. panning of the phage displayed antibody library. This is described in Methods 36, 43-60 (2005).
  • epitope binding domain refers to a domain which is able to specifically recognize and bind an antigenic epitope.
  • the classic example of an epitope binding domain would be an antibody paratope comprising a V H domain and a V L domain forming an antigen binding site.
  • the sequences of the antibody heavy chain variable regions and/or the light chain variable regions disclosed herein may be modified by, for example, insertions, substitutions and/or deletions to the extent that the epitope binding domain maintains the ability to bind to the cognate antigen.
  • the skilled person can ascertain the maintenance of this activity by performing the functional assays described herein, or known in the art.
  • the heavy chain variable region comprises no more than 20 insertions, substitutions and/or deletions, such as no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 insertion, substitution and/or deletion.
  • the light chain variable region comprises no more than 20 insertions, substitutions and/or deletions, such as no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 insertion, substitution and/or deletion.
  • the antibodies of the disclosure include comprising VH and VL domains with amino acid sequences that are identical to the sequences described herein.
  • therapeutic index is used as a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes death (in animal studies) or toxicity (in human studies).
  • LD lethal dose for 50% of the population
  • TD toxic dose for 50% of the population
  • ED minimum effective dose for 50% of the population.
  • the levels of “effective” and “toxic” doses can be readily determined by a medical practitioner or person skilled in the art. When comparing the therapeutic indexes of the site-specific and non-site-specific conjugates, the levels of “effective” and “toxic” are determined in an identical manner
  • non site-specific conjugate refers to a conjugate which is identical to the defined or claimed site-specific conjugate in all respects apart from the position(s) at which the Drug units (D L ) are conjugated to antibody heavy chain constant region, or a portion thereof.
  • Drug units (D L ) are uniformly and consistently conjugated to the specified residue(s)
  • the degree and position of conjugation of Drug unit (D L ) to the antibody is variable from batch to batch.
  • a site specific antibody-drug conjugate of the disclosure there are two Drug units (D L ), one conjugated to each of the position 442 residues (kabat numbering) of the two antibody heavy chain constant regions, or a portions thereof.
  • the ‘otherwise identical non site-specific conjugate’ for this example would be an antibody with identical amino acid sequence and polypeptide structure, also with two conjugated Drug unit (D L ); however, the Drug unist (D L ) would not uniformly and consistently conjugated to each 442 position, but rather conjugated to a selection of different positions the precise combination of which varies from conjugate to conjugate within a population (for example, conjugation may be via lysine side chains or by reduced interchain disulfide bonds).
  • properties such as affinity, therapeutic index and stability are bulk properties measured at a population level, as opposed to being measured at a molecular level.
  • the comparisons made herein between the properties of a site-specific conjugate and an “otherwise identical non site-specific conjugate” are comparisons of properties exhibited by populations of those molecules.
  • the humanised antibody of the disclosure may be conjugated to a functional moiety.
  • functional moieties include an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a reporter (such as a fluorophore, a chromophore, or a dye), a toxin, a hapten, an enzyme, a binding member (such as an antibody, or an antibody fragment), a radioisotope, solid matrixes, semisolid matrixes and combinations thereof, or an organic moiety.
  • Examples of a drug include a cytotoxic agent, a chemotherapeutic agent, a peptide, a peptidomimetic, a protein scaffold, DNA, RNA, siRNA, microRNA, and a peptidonucleic acid.
  • the functional moiety is a PBD drug moiety.
  • the humanised antibody is conjugated to a therapeutic agent or drug moiety that modifies a given biological response.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF- ⁇ , TNF- ⁇ , AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No.
  • a thrombotic agent or an anti-angiogenic agent e.g., angiostatin or endostatin
  • a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 (“IL-I”), interleukin-2 (“IL-2”), interleukin-4 (“IL-4”), interleukin-6 (“IL-6”), interleukin-7 (“IL-7”), interleukin-9 (“IL-9”), interleukin-15 (“IL-15”), interleukin-12 (“IL-12”), granulocyte macrophage colony stimulating factor (“GMCSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g.,growth hormone (“GH”)).
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • IL-7 interleukin-7
  • IL-9 interleukin-9
  • IL-15 interleuk
  • Examples of a reporter include a fluorophore, a chromophore, a radionuclide, and an enzyme.
  • a reporter include a fluorophore, a chromophore, a radionuclide, and an enzyme.
  • Such antibody-reporter conjugates can be useful for monitoring or prognosing the development or progression of a disorder (such as, but not limited to cancer) as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can accomplished by fusing or conjugating the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), cobalt ( 57 Co),
  • binding member examples include an antibody or antibody fragment, and biotin and/or streptavidin.
  • a toxin, cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • toxins include radioisotopes such as 131 I, a ribosome inactivating protein such as pseudomonas exotoxin (PE38 fragment), plant or bacterial toxins such as ricin, the a-chain of ricin, saporin, pokeweed antiviral protein, diphtheria toxin, or Pseudomonas exotoxin A (Kreitman and Pastan (1998) Adv. Drug Delivery Rev. 31:53.).
  • toxins and cytotoxins include, e.g., a cytostatic or cytocidal agent, or a radioactive metal ion, e.g., alpha-emitters.
  • examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homo logs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, c
  • Chemical toxins can also be taken from the group chosen from duocarmycin (U.S. Pat. Nos. 5,703,080; 4,923,990), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatinum, etoposide, bleomycin and 5-fluorouracil.
  • chemotherapeutic agents also include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate,
  • the cytotoxic agent is chosen from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • the cytotoxic agent is paclitaxel, docetaxel, CC-I 065, SN-3 8, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, DM-I, an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), eleutherobin or netropsin.
  • auristatin E or auristatin F such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), eleutherobin or
  • the cytoxic agent is Maytansine or Maytansinoids, and derivatives thereof, wherein an antibodies (full length or fragments) of the disclosure are conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization.
  • the toxin is a small molecule or protein toxins, such as, but not limited to abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustralin, ocratoxin A, patulin,
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • the humanized antibody may be modified by conjugation to an organic moiety. Such modification can produce an antibody or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life).
  • 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 cytotoxic or cytostatic agent is a dolastatin. In more specific embodiments, the dolastatin is of the auristatin class.
  • the cytotoxic or cytostatic agent is MMAE.
  • the cytotoxic or cytostatic agent is AEFP.
  • the cytotoxic or cytostatic agent is MMAF.
  • the humanized antibody 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.
  • a molecular weight of about 800 to about 150,000 Daltons for example PEG5000 and PEG20,000, wherein the numerical component of the name 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 N,N-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 N,N-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 (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis- ⁇ 9-octadecanoate (C18, oleate), all cis- ⁇ 5,8,11,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecane
  • a “modifying agent” refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group
  • aAn “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, Hernanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)).
  • 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.
  • Suitable linker moieties include, for example, tetraethylene glycol, —(CH2)3-, —NH—(CH2)6-NH—, —(CH2)2-NH— and —CH2-O—CH2-CH2-O—CH2-CH2-O—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 above conjugates 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., inter-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., 5:411-417 (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., 5:
  • 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 R 2 + , NHR 3 + , NR 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as 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.
  • C 1-12 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).
  • C 1-4 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 (C l ), 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 (C 1 ), 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 ).
  • C 2-12 Alkenyl The term “C 2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
  • unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH ⁇ CH 2 ), 1-propenyl (—CH ⁇ CH—CH 3 ), 2-propenyl (allyl, —CH—CH ⁇ CH 2 ), isopropenyl (1-methylvinyl, —C(CH 3 ) ⁇ CH 2 ), butenyl (C 4 ), pentenyl (C 5 ), and hexenyl (C 6 ).
  • C 2-12 alkynyl The term “C 2-12 alkynyl” as used herein, pertains 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).
  • C 3-12 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:
  • C 3-20 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. C 3-20 , C 3-7 , C 5-6 , etc.
  • the term “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 1 aziridine (C 3 ), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C 5 ), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C 5 ), piperidine (C 6 ), dihydropyridine (C 6 ), tetrahydropyridine (C 6 ), azepine (C 7 );
  • O 1 oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (C 5 ), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 );
  • N 2 imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline (dihydropyrazole) (C 5 ), piperazine (C 6 );
  • N 1 O 1 tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 );
  • N 1 S 1 thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 );
  • O 1 S 1 oxathiole (C 5 ) and oxathiane (thioxane) (C 6 ); and,
  • N 1 O 1 S 1 oxathiazine (C 6 ).
  • 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, galactopyranose, and talopyranose.
  • furanoses C 5
  • arabinofuranose such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse
  • pyranoses C 6
  • allopyranose altropyranose
  • glucopyranose glucopyranose
  • mannopyranose gulopyranose
  • idopyranose galactopyranose
  • C 5-20 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 3 to 20 ring atoms.
  • C 5-7 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 7 ring atoms and the term “C 5-10 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 5 to 10 ring atoms.
  • each ring has from 5 to 7 ring atoms.
  • the prefixes e.g. C 3-20 , C 5-7 , C 5-6 , C 5-10 , etc.
  • the term “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 (C 10 ), azulene (C 10 ), anthracene (C 14 ), phenanthrene (C 14 ), naphthacene (C 18 ), and pyrene (C 16 ).
  • benzene i.e. phenyl
  • C 10 naphthalene
  • azulene C 10
  • anthracene C 14
  • phenanthrene C 14
  • naphthacene C 18
  • pyrene C 16
  • aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C 9 ), indene (C 9 ), isoindene (C 9 ), tetraline (1,2,3,4-tetrahydronaphthalene (C 10 ), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), and aceanthrene (C 16 ).
  • indane e.g. 2,3-dihydro-1H-indene
  • indene C 9
  • isoindene C 9
  • tetraline (1,2,3,4-tetrahydronaphthalene C 10
  • acenaphthene C 12
  • fluorene C 13
  • phenalene C 13
  • acephenanthrene C 15
  • aceanthrene
  • the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”.
  • heteroaryl groups include, but are not limited to, those derived from:
  • N 1 pyrrole (azole) (C 5 ), pyridine (azine) (C 6 );
  • N 1 O 1 oxazole (C 5 ), isoxazole (C 5 ), isoxazine (C 6 );
  • N 1 S 1 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 );
  • heteroaryl which comprise fused rings, include, but are not limited to:
  • C 13 (with 3 fused rings) derived from carbazole (N 1 ), dibenzofuran (O 1 ), dibenzothiophene (S 1 ), carboline (N 2 ), perimidine (N 2 ), pyridoindole (N 2 ); and,
  • C 14 (with 3 fused rings) derived from acridine (N 1 ), xanthene (O 1 ), thioxanthene (S 1 ), oxanthrene (O 2 ), phenoxathiin (O 1 S 1 ), phenazine (N 2 ), phenoxazine (N 1 O 1 ), phenothiazine (N 1 S 1 ), thianthrene (S 2 ), phenanthridine (N 1 ), phenanthroline (N 2 ), phenazine (N 2 ).
  • Halo —F, —Cl, —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 5-20 aryl group (also referred to as a C 5-20 aryloxy group), preferably a C 1-7 alkyl group.
  • 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 5-20 aryl group (also referred to as a C 5-20 aryloxy group), preferably a C 1-7 alkyl group.
  • Alkoxy —OR, wherein R is an alkyl group, for example, a C 1-7 alkyl group.
  • C 1-7 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) (isobutoxy), and —O(tBu) (tert-butoxy).
  • Acetal —CH(OR 1 )(OR 2 ), wherein R 1 and R 2 are independently acetal substituents, 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, 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.
  • Examples of 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 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a C 1-7 alkyl group.
  • R 1 is a hemiacetal 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.
  • 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 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.
  • Examples 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).
  • Hemiketal —CR(OH)(OR 1 ), where R 1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, 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.
  • hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).
  • Imino (imine): ⁇ NR wherein R is an imino substituent, for example, hydrogen, 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.
  • ester groups include, but are not limited to, ⁇ NH, ⁇ NMe, ⁇ NEt, and ⁇ NPh.
  • R is an acyl substituent, for example, a C 1-7 alkyl group (also referred to as C 1-7 alkylacyl or C 1-7 alkanoyl), a C 3-20 heterocyclyl group (also referred to as C 3-20 heterocyclylacyl), or a C 5-20 aryl group (also referred to as C 5-20 arylacyl), preferably a C 1-7 alkyl group.
  • R is an acyl substituent, for example, a C 1-7 alkyl group (also referred to as C 1-7 alkylacyl or C 1-7 alkanoyl), a C 3-20 heterocyclyl group (also referred to as C 3-20 heterocyclylacyl), or a C 5-20 aryl group (also referred to as C 5-20 arylacyl), preferably a C 1-7 alkyl group.
  • acyl groups include, but are not limited to, —C( ⁇ O)CH 3 (acetyl), —C( ⁇ O)CH 2 CH 3 (propionyl), —C( ⁇ O)C(CH 3 ) 3 (t-butyryl), and —C( ⁇ O)Ph (benzoyl, phenone).
  • Thiolocarboxy thiolocarboxylic acid: —C( ⁇ O)SH.
  • Imidic acid —C( ⁇ NH)OH.
  • Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C( ⁇ O)OR, wherein R is an ester 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.
  • ester groups include, but are not limited to, —C( ⁇ O)OCH 3 , —C( ⁇ O)OCH 2 CH 3 , —C( ⁇ O)OC(CH 3 ) 3 , and —C( ⁇ O)OPh.
  • R is an acyloxy 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.
  • acyloxy groups include, but are not limited to, —OC( ⁇ O)CH 3 (acetoxy), —OC( ⁇ O)CH 2 CH 3 , —OC( ⁇ O)C(CH 3 ) 3 , —OC( ⁇ O)Ph, and —OC( ⁇ O)CH 2 Ph.
  • Oxycarboyloxy —OC( ⁇ O)OR, wherein R is an ester 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.
  • ester groups include, but are not limited to, —OC( ⁇ O)OCH 3 , —OC( ⁇ O)0CH 2 CH 3 , —OC( ⁇ O)OC(CH 3 ) 3 , and —OC( ⁇ O)OPh.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C 1-7 alkyl group (also referred to as C 1-7 alkylamino or di-C 1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 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.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C 1-7 alkyl group (also referred to as C 1-7 alkylamino or di-C 1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group, or, in the case of a “cyclic” amino group, R 1 and R 2 ,
  • 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 , —NHCH 3 , —NHC(CH 3 ) 2 , —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , and —NHPh.
  • Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
  • amido groups include, but are not limited to, —C( ⁇ O)NH 2 , —C( ⁇ O)NHCH 3 , —C( ⁇ O)N(CH 3 ) 2 , —C( ⁇ O)NHCH 2 CH 3 , and —C( ⁇ O)N(CH 2 CH 3 ) 2 , as well as amido groups in which R 1 and R 2 , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
  • Thioamido (thiocarbamyl) —C( ⁇ S)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • amido groups include, but are not limited to, —C( ⁇ S)NH 2 , —C( ⁇ S)NHCH 3 , —C( ⁇ S)N(CH 3 ) 2 , and —C( ⁇ S)NHCH 2 CH 3 .
  • acylamide groups include, but are not limited to, —NHC( ⁇ O)CH 3 , —NHC( ⁇ O)CH 2 CH 3 , and —NHC( ⁇ O)Ph.
  • R 1 and R 2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
  • Aminocarbonyloxy —OC( ⁇ O)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • Examples of aminocarbonyloxy groups include, but are not limited to, —OC( ⁇ O)NH 2 , —OC( ⁇ O)NHMe, —OC( ⁇ O)NMe 2 , and —OC( ⁇ O)NEt 2 .
  • 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 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.
  • 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.
  • imino groups include, but are not limited to, ⁇ NH, ⁇ NMe, and ⁇ NEt.
  • amidine groups include, but are not limited to, —C( ⁇ NH)NH 2 , —C( ⁇ NH)NMe 2 , and —C( ⁇ NMe)NMe 2 .
  • Nitroso —NO.
  • C 1-7 alkylthio groups include, but are not limited to, —SCH 3 and —SCH 2 CH 3 .
  • Disulfide —SS—R, wherein R is a disulfide 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 (also referred to herein as C 1-7 alkyl disulfide).
  • R is a disulfide 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 (also referred to herein as C 1-7 alkyl disulfide).
  • C 1-7 alkyl disulfide groups include, but are not limited to, —SSCH 3 and —SSCH 2 CH 3 .
  • R is a sulfine 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.
  • sulfine groups include, but are not limited to, —S( ⁇ O)CH 3 and —S( ⁇ O)CH 2 CH 3 .
  • sulfone groups include, but are not limited to, —S( ⁇ O) 2 CH 3 (methanesulfonyl, mesyl), —S( ⁇ O) 2 CF 3 (triflyl), —S( ⁇ O) 2 CH 2 CH 3 (esyl), —S( ⁇ O) 2 C 4 F 9 (nonaflyl), —S( ⁇ O) 2 CH 2 CF 3 (tresyl), —S( ⁇ O) 2 CH 2 CH 2 NH 2 (tauryl), —S( ⁇ O) 2 Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen
  • Sulfinate (sulfinic acid ester): —S( ⁇ O)OR; wherein R is a sulfinate 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 is a sulfinate 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.
  • sulfinate groups include, but are not limited to, —S( ⁇ O)OCH 3 (methoxysulfinyl; methyl sulfinate) and —S( ⁇ O)OCH 2 CH 3 (ethoxysulfinyl; ethyl sulfinate).
  • Sulfonate (sulfonic acid ester): —S( ⁇ O) 2 OR, wherein R is a sulfonate 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 is a sulfonate 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.
  • sulfonate groups include, but are not limited to, —S( ⁇ O) 2 OCH 3 (methoxysulfonyl; methyl sulfonate) and —S( ⁇ O) 2 OCH 2 CH 3 (ethoxysulfonyl; ethyl sulfonate).
  • Sulfinyloxy —OS( ⁇ O)R, wherein R is a sulfinyloxy 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 is a sulfinyloxy 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.
  • sulfinyloxy groups include, but are not limited to, —OS( ⁇ O)CH 3 and —OS( ⁇ O)CH 2 CH 3 .
  • Sulfonyloxy —OS( ⁇ O) 2 R, wherein R is a sulfonyloxy 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 is a sulfonyloxy 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.
  • sulfonyloxy groups include, but are not limited to, —OS( ⁇ O) 2 CH 3 (mesylate) and —OS( ⁇ O) 2 CH 2 CH 3 (esylate).
  • Sulfate —OS( ⁇ O) 2 OR; wherein 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 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.
  • sulfate groups include, but are not limited to, —OS( ⁇ O) 2 OCH 3 and —SO( ⁇ O) 2 OCH 2 CH 3 .
  • Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S( ⁇ O)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • sulfamyl groups include, but are not limited to, —S( ⁇ O)NH 2 , —S( ⁇ O)NH(CH 3 ), —S( ⁇ O)N(CH 3 ) 2 , —S( ⁇ O)NH(CH 2 CH 3 ), —S( ⁇ O)N(CH 2 CH 3 ) 2 , and —S( ⁇ O)NHPh.
  • Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S( ⁇ O) 2 NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • sulfonamido groups include, but are not limited to, —S( ⁇ O) 2 NH 2 , —S( ⁇ O) 2 NH(CH 3 ), —S( ⁇ O) 2 N(CH 3 ) 2 , —S( ⁇ O) 2 NH(CH 2 CH 3 ), —S( ⁇ O) 2 N(CH 2 CH 3 ) 2 , and —S( ⁇ O) 2 NHPh.
  • Sulfamino —NR 1 S( ⁇ O) 2 OH, wherein R 1 is an amino substituent, as defined for amino groups.
  • R 1 is an amino substituent, as defined for amino groups.
  • sulfamino groups include, but are not limited to, —NHS( ⁇ O) 2 OH and —N(CH 3 )S( ⁇ O) 2 OH.
  • Sulfonamino —NR 1 S( ⁇ O) 2 R, wherein R 1 is an amino substituent, as defined for amino groups, and R is a sulfonamino 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 is an amino substituent, as defined for amino groups
  • R is a sulfonamino 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.
  • sulfonamino groups include, but are not limited to, —NHS( ⁇ O) 2 CH 3 and —N(CH 3 )S( ⁇ O) 2 C 6 H 5 .
  • Sulfinamino —NR 1 S( ⁇ O)R, wherein R 1 is an amino substituent, as defined for amino groups, and R is a sulfinamino 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 is an amino substituent, as defined for amino groups
  • R is a sulfinamino 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.
  • sulfinamino groups include, but are not limited to, —NHS( ⁇ O)CH 3 and —N(CH 3 )S( ⁇ O)C 6 H 5 .
  • R is a phosphino 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.
  • 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 .
  • Phospho —P( ⁇ O) 2 .
  • Phosphinyl phosphine oxide: —P( ⁇ O)R 2 , wherein R is a phosphinyl 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 or a C 5-20 aryl group.
  • R is a phosphinyl 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 or a C 5-20 aryl group.
  • Examples of phosphinyl groups include, but are not limited to, —P( ⁇ O)(CH 3 ) 2 , —P( ⁇ O)(CH 2 CH 3 ) 2 , —P( ⁇ O)(t-Bu) 2 , and —P( ⁇ O)(Ph) 2 .
  • Phosphonic acid (phosphono) —P( ⁇ O)(OH) 2 .
  • R is a phosphonate 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.
  • Examples of phosphonate groups include, but are not limited to, —P( ⁇ O)(OCH 3 ) 2 , —P( ⁇ O)(OCH 2 CH 3 ) 2 , —P( ⁇ O)(O-t-Bu) 2 , and —P(
  • Phosphoric acid —OP( ⁇ O)(OH) 2 .
  • Phosphate (phosphonooxy ester) —OP( ⁇ O)(OR) 2
  • R is a phosphate 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.
  • phosphate groups include, but are not limited to, —OP( ⁇ O)(OCH 3 ) 2 , —OP( ⁇ O)(OCH 2 CH 3 ) 2 , —OP( ⁇ O)(O-t-Bu) 2 , and —OP( ⁇ O)(OPh) 2 .
  • Phosphorous acid —OP(OH) 2 .
  • Phosphite —OP(OR) 2 , where 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 , —OP(OCH 2 CH 3 ) 2 , —OP(O-t-Bu) 2 , and —OP(OPh) 2 .
  • Phosphoramidite —OP(OR 1 )—NR 2 2 , where R 1 and R 2 are phosphoramidite substituents, for example, —H, a (optionally substituted) 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.
  • Examples of phosphoramidite groups include, but are not limited to, —OP(OCH 2 CH 3 )—N(CH 3 ) 2 , —OP(OCH 2 CH 3 )—N(i-Pr) 2 , and —OP(OCH 2 CH 2 CN)—N(i-Pr) 2 .
  • Phosphoramidate —OP( ⁇ O)(OR 1 )—NR 2 2 , where R 1 and R 2 are phosphoramidate substituents, for example, —H, a (optionally substituted) 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.
  • Examples of phosphoramidate groups include, but are not limited to, —OP( ⁇ O)(OCH 2 CH 3 )—N(CH 3 ) 2 , —OP( ⁇ O)(OCH 2 CH 3 )—N(i-Pr) 2 , and —OP( ⁇ O)(OCH 2 CH 2 CN)—N(i-Pr) 2 .
  • C 3-12 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.
  • linear saturated C 3-12 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), —CH 2 CH 2 CH 2 CH 2 — (butylene), —CH 2 CH 2 CH 2 CH 2 CH 2 — (pentylene) and —CH 2 CH 2 CH 2 CH— 2 CH 2 CH 2 CH 2 — (heptylene).
  • Examples of branched saturated C 3-12 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 CH 2 —, —CH 2 CH(CH 3 )CH 2 —, —CH 2 CH(CH 3 )CH 2 CH 2 —, —CH(CH 2 CH 3 )—, —CH(CH 2 CH 3 )CH 2 —, and —CH 2 CH(CH 2 CH 3 )CH 2 —.
  • linear partially unsaturated C 3-12 alkylene groups include, but are not limited to, —CH ⁇ CH—CH 2 —, —CH 2 —CH ⁇ CH 2 —, —CH ⁇ CH—CH 2 —CH 2 —, —CH ⁇ CH—CH 2 —CH 2 —, —CH ⁇ CH—CH ⁇ CH—, —CH ⁇ CH—CH ⁇ CH—OH 2 —, —CH ⁇ CH—CH ⁇ CH—CH 2 —CH 2 —, —CH ⁇ CH—CH 2 —CH ⁇ CH—, —CH ⁇ CH—CH 2 —CH 2 —CH ⁇ CH—, and —CH 2 —C ⁇ C—CH 2 —.
  • Examples of branched partially unsaturated C 3-12 alkylene groups include, but are not limited to, —C(CH 3 ) ⁇ CH—, —C(CH 3 ) ⁇ CH—CH 2 —, —CH ⁇ CH—CH(CH 3 )— and —C ⁇ C—CH(CH 3 )—.
  • C 3-12 cycloalkylenes examples include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
  • C 3-12 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).
  • 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 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:
  • R′ 10 is R as defined above.
  • 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 pertains to a group having the following structure:
  • R′ 10 is R as defined above.
  • suitable groups are described on pages 633 to 647 as amide protecting groups 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.
  • the groups Carbamate nitrogen protecting group and Hemi-aminal nitrogen protecting group may be jointly termed a “nitrogen protecting group for synthesis”.
  • the present disclosure 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 L1′ or R L2′ removed.
  • the present disclosure 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.
  • a cysteine thiol, or an amine e.g.
  • 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 R L1 or R L2 .
  • 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 separation/analysis technique LC/MS.
  • 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) Bioconjugate Techniques; Academic Press: New York, p 234-242).
  • 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 group:
  • Ab is the antibody (L)
  • L 1 is a linker
  • A is a connecting group connecting L 1 to the antibody
  • L 2 is a covalent bond or together with —OC( ⁇ O)— forms a self-immolative linker
  • 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 disclosure.
  • 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.
  • L 2 is present and together with —C( ⁇ O)O— forms a self-immolative linker. In one embodiment, L 2 is a substrate for enzymatic activity, thereby allowing release of L-R L′ from the N10 position.
  • 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
  • —C( ⁇ O)O— and L 2 together form the group:
  • Y is NH
  • n is 0 or 1. Preferably, n is 0.
  • the self-immolative linker may be referred to as a p-aminobenzylcarbonyl linker (PABC).
  • PABC p-aminobenzylcarbonyl linker
  • the group L* is a linker L 1 as described herein, which may include a dipeptide group.
  • —C( ⁇ O)O— and L 2 together form a group selected from:
  • —C( ⁇ O)O— and L 2 together form a group selected from:
  • 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 1 —X 2 —CO—, where —NH— and —CO— represent the N- and C-terminals of the amino acid groups X 1 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 functionality.
  • the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO— is selected from:
  • Cit is citrulline
  • the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO— is selected from:
  • the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —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
  • 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.
  • 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, Ile, 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 —X 2 —CO—NH—.
  • —NH—X 1 — is connected directly to A.
  • A may comprise the functionality —CO— thereby to form an amide link with —X 1 —.
  • L 1 and L 2 together with —OC( ⁇ O)— comprise the group NH—X 1 —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 self-immolative linker and the dipeptide together form the group —NH-Val-Ala-CO—NH—PABC—, which is illustrated below:
  • the self-immolative linker and the dipeptide together form the group —NH-Val-Cit-CO—NH—PABC—, which is illustrated below:
  • 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.
  • 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.
  • L 2 together with —OC( ⁇ O)— represents:
  • 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 —NO 2 or glucoronic acid.
  • the former may be susceptible to the action of a nitroreductase, the latter to the action of a ⁇ -glucoronidase.
  • 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
  • Y is —NH— or —C( ⁇ O)—, thereby to form an amide bond between L 1 and Y.
  • 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:
  • the group A is:
  • the group A is:
  • the group A is:
  • connection between the antibody and A is through a thiol residue of the antibody and a maleimide group of A.
  • connection between the antibody and A is:
  • 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 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:
  • N ⁇ or O- are part of D.
  • 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 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO— is selected from:
  • Cit is citrulline.
  • —NH— is the amino group of X 1
  • CO is the carbonyl group of X 2 .
  • the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO— is selected from:
  • the group —X 1 —X 2 — in dipeptide, —NH—X 1 —X 2 —CO—, is -Phe-Lys- or -Val-Ala-.
  • dipeptide combinations of interest include:
  • dipeptide combinations may be used, including those described above.
  • L 1 - D is:
  • 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:
  • the groups A-L 1 are:
  • the groups A-L 1 are:
  • the groups A-L 1 are:
  • the groups A-L 1 are:
  • the groups A-L 1 are:
  • the groups A-L 1 are:
  • the groups A-L 1 is:
  • the groups A-L 1 are:
  • the group A-L 1 are:
  • the groups A 1 -L 1 are:
  • the groups A 1 -L 1 are:
  • the groups A 1 -L 1 are:
  • the groups A 1 -L 1 are:
  • the groups A 1 -L 1 are:
  • the groups A 1 -L 1 are:
  • 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. Lübke, 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.
  • Amino acid side chains include hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH 2 OH, —CH(OH)CH 3 , —CH 2 CH 2 SCH 3 , —CH 2 CONH 2 , —CH 2 COOH, —CH 2 CH 2 CONH 2 , —CH 2 CH 2 COOH, —(CH 2 ) 3 NHC( ⁇ NH)NH 2 , —(CH 2 ) 3 NH 2 , —(CH 2 ) 3 NHCOCH 3 , —(CH 2 ) 3 NHCHO, —(CH 2 ) 4 NHC( ⁇ NH)NH 2 , —(CH 2 ) 4 NH 2 , —(CH 2 ) 4 NHCOCH 3 , —(CH 2 )
  • 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 diastereomeric.
  • 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 C 1 -C 8 alkyl, —O—(C 1 —C 8 alkyl), -halogen, —NO 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 C 1 -C 8 alkyl, —O—(C 1 -C 8 alkyl), -halogen, —NO 2 or —CN
  • m is an integer ranging from 0-4.
  • the “self-immolative linker” PAB para-aminobenzyloxycarbonyl
  • PAB para-aminobenzyloxycarbonyl
  • valine-citrulline dipeptide PAB analog reagent has a 2,6 dimethyl phenyl group and has the structure:
  • Linker reagents useful for the antibody drug conjugates of the disclosure 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 1,11-bis-maleimidotriethyleneglycol (BM(PEO) 3 ), which are commercially available from Pierce Bio
  • 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 HCl
  • 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 Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in accordance with procedures described in Toki et al (2002) J. Org. Chem. 67:1866-1872; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
  • 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 2006/116422; 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.
  • an antibody bears only one reactive cysteine thiol group, a multitude of drug moieties may be attached through a dendritic or branched linker.
  • the conjugate of the first aspect of the disclosure may have a capping group R C at the N10 position.
  • the group R C is removable from the N10 position of the PBD moiety to leave an N10-C11 imine bond, a carbinolamine, a substituted carbinolamine, where QR 11 is OSO 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-C11 imine bond, a carbinolamine, a substituted cabinolamine, or, where QR 11 is OSO 3 M, a bisulfite adduct. In one embodiment, R C is a protecting group that is removable to leave an N10-C11 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-C11 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 .
  • Compounds having a 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. In this embodiment, 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-C11 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.
  • R C is a group:
  • G 2 and OC( ⁇ O) together form a carbamate protecting group as defined above.
  • L 1 is as defined above in relation to R 10 .
  • L 2 is as defined above in relation to R 10 .

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TW202330608A (zh) 2021-09-30 2023-08-01 大陸商江蘇恆瑞醫藥股份有限公司 吡咯并苯并二氮雜卓類衍生物及其偶聯物、其製備方法及其應用

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