[go: up one dir, main page]

WO2017137556A1 - Conjugués anticorps anti-her2-pyrrolobenzodiazépine - Google Patents

Conjugués anticorps anti-her2-pyrrolobenzodiazépine Download PDF

Info

Publication number
WO2017137556A1
WO2017137556A1 PCT/EP2017/052991 EP2017052991W WO2017137556A1 WO 2017137556 A1 WO2017137556 A1 WO 2017137556A1 EP 2017052991 W EP2017052991 W EP 2017052991W WO 2017137556 A1 WO2017137556 A1 WO 2017137556A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cysteine
amino acid
antibody
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2017/052991
Other languages
English (en)
Inventor
Patricius Hendrikus Cornelis VAN BERKEL
Philip Wilson Howard
Elizabeth DUNNY
Ian Hutchinson
Luke Masterson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ADC Therapeutics SA
MedImmune Ltd
Original Assignee
ADC Therapeutics SA
MedImmune Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ADC Therapeutics SA, MedImmune Ltd filed Critical ADC Therapeutics SA
Publication of WO2017137556A1 publication Critical patent/WO2017137556A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to conjugates comprising an antibody which binds HER2 and a specific pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • PBDs pyrrolobenzodiazepines
  • the PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C, et al, Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity.
  • sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C, et al, Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity.
  • -136 One example of -136):
  • Dimeric PBD compounds bearing C2 aryl substituents, such as SG2202 (ZC-207), are disclosed in WO 2005/085251 :
  • PBD dimers which are more potent than SG2000, have been developed, including SG2057 and SG2202. They exhibit picomolar/sub-picomolar activity against a range of human tumour cell lines and demonstrate curative activity in human tumour xenograft models. making reference to:
  • SG2057 has the structure:
  • claim 54 of this application includes the formula:
  • n is from 1 to 24, more preferably 4 to 8.
  • Claim 54 of this application also includes the formula:
  • n is from 1 to 24, more preferably 4 to 8.
  • WO 201 1/130598 also discloses antibody-drug conjugates including these drug linkers, for example 1 10 (antiSteap1 -15d), example 1 14 (tastuzumab-15d) and example 1 15
  • WO 2014/057074 discloses:
  • WO2015/052322 discloses:
  • the present inventors have surprisingly found that although SG2000 is at least 10 times less cytotoxic than SG2057 (see Hartley et al 2012), particular antibody-drug conjugates appear to show at least comparable activity. These conjugates have been shown to have surprisingly well tolerated in toxicity studies in a variety of species. This leads to the conjugates exhibiting high therapeutic indices and thus are promising clinical candidates.
  • the present invention provides Conjugates of formula I :
  • L is a Li and unit (i.e., a targeting agent), D is a Drug Linker unit of formula A:
  • the Ligand unit is an antibody which binds HER2 or an antigen-binding fragment of an antibody which binds HER2.
  • the antibody may comprise an amino acid substitution of an interchain cysteine residue by an amino acid that is not cysteine, wherein the conjugation of the drug moiety to the antibody is at an interchain cysteine residue.
  • ADC antibody-drug conjugate
  • the present disclosure provides an antibody-drug conjugate (ADC) comprising a Drug Linker unit of formula A conjugated to an antibody which binds HER2 and which comprises an amino acid substitution of an interchain cysteine residue by an amino acid that is not cysteine wherein the conjugation of the drug moiety to the antibody is at an interchain cysteine residue.
  • the antibodies described herein may comprise one or more substitution of an interchain cysteine residue 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 N0.1 10, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 is substituted by an amino acid that is not cysteine;
  • 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 103 of SEQ ID NO.1 10.
  • cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 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;
  • each of the cysteines at positions 103, 106, and 109 in SEQ ID NO: 120 is substituted by an amino acid that is not cysteine; and wherein the 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 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;
  • each of the cysteines at positions 14, 106, and 109 in SEQ ID NO: 120 is substituted by an amino acid that is not cysteine;
  • 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 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.
  • 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.130, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • SEQ ID NO: 130 is substituted by an amino acid that is not cysteine; and wherein the 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 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;
  • each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is substituted by an amino acid that is not cysteine;
  • 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.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.
  • a second aspect of the present invention provides the use of a conjugate of the first aspect of the invention in the manufacture of a medicament for treating a proliferative disease.
  • the second aspect also provides a conjugate of the first aspect of the invention for use in the treatment of a proliferative disease.
  • the third aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the invention to a patient in need thereof.
  • proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • proliferative conditions include, but are not limited to cancers. Cancers of particular interest include, but are not limited to breast cancers, gastric cancers, bladder cancers, prostate cancers, leukemias and ovarian cancers.
  • Figure 1 shows the effect on the volume of a BT-474 xenograft tumour following treatment with a conjugate of the present invention and with a comparative conjugate;
  • Figure 2 shows the effect on the volume of a BT-474 xenograft following treatment with a conjugate of the present invention and with a comparative conjugate at a higher dose;
  • Figure 3 shows the effect on the volume of HER2 (1 +) breast cancer following treatment with a conjugate of the present invention and with a comparative conjugate at two doses
  • Figure 4 shows the effect on the volume of HER2 (2+) breast cancer following treatment with a conjugate of the present invention at two doses and with a comparative conjugate at a single dose.
  • the antibodies described herein may comprise one or more substitution of an interchain cysteine residue by an amino acid that is not cysteine.
  • the antibodies described herein retains at least one unsubstituted interchain cysteine residue for conjugation of the drug moiety to the antibody.
  • the antibody has an even integral number of interchain cysteine residues (e.g., at least two, four, six or eight).
  • Naturally occurring antibodies typically 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 ⁇ -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 lgG1 isotype heavy chain ⁇ ' - 220, 226, and 229 in the EU index set forth in Kabat
  • cysteine in each light chain 'LC - K(kappa)214 or A(lambda)213
  • interchain cysteines 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 CHi domain of the heavy chain, and in the hinge region.
  • the number of interchain cysteine residues in an antibody depends on the antibody isotype.
  • 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 described herein may 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.
  • the phrase “...a light chain comprising the amino acid sequence of SEQ ID NO. XXX wherein the cysteine at position YYY in SEQ ID NO: XXX, is substituted by an amino acid that is not cysteine. " Has the same meaning as "...a light chain comprising the amino acid sequence of SEQ ID NO. XXX wherein the cysteine at position YYY in SEQ ID NO: XXX, is deleted.
  • 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.
  • the antibody of the conjugates described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO.1 10, or fragment thereof, wherein each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10, if present, is substituted by an amino acid that is not cysteine.
  • SEQ ID NO: 1 13 discloses a heavy chain comprising the amino acid sequence of SEQ ID NO.1 10 wherein each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 is substituted by a serine residue.
  • SEQ ID NO: 1 14 discloses a heavy chain comprising the amino acid sequence of SEQ ID N0.1 10 wherein each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 is substituted by a valine residue.
  • the drug moiety is conjugated to the cysteine at position 103 of SEQ ID NO.1 10.
  • 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 1 1 1 , 1 14, 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.
  • all but one of the cysteines at positions 1 1 1 1 , 1 14, 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 1 1 1 , 1 14, 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 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 N0.1 10, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 is substituted by an amino acid that is not cysteine;
  • 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.
  • cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 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 drug moiety is conjugated to the cysteine at position 103 of SEQ ID N0.1 10.
  • 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;
  • each of the cysteines at positions 103, 106, and 109 in SEQ ID NO: 120 is substituted by an amino acid that is not cysteine;
  • 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 cysteine at position 102 in SEQ ID NO: 120 is also substituted by an amino acid that is not cysteine.
  • the 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 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.120, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • each of the cysteines at positions 14, 106, and 109 in SEQ ID NO: 120 is substituted by an amino acid that is not cysteine;
  • 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 cysteine at position 102 in SEQ ID NO: 120 is also substituted by an amino acid that is not cysteine.
  • the cysteines at positions 14, 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 drug moiety is conjugated to the cysteine at position 103 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, and a light chain comprising the amino acid sequence of SEQ ID NO. 150 or SEQ ID NO. 160;
  • each of the cysteines at positions 1 1 1 1 , 1 14, 120, 126, 129, 135, 141 , 144, 150, 156, and 159 in SEQ ID NO: 130 is substituted by an amino acid that is not cysteine; and wherein the 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 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 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;
  • each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 is substituted by an amino acid that is not cysteine;
  • 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.
  • each of the cysteines at positions 106 and 109 in SEQ ID NO: 140 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.
  • drug moiety is conjugated to the cysteine at position 14 of SEQ ID NO.140.
  • the antibody of the conjugates described herein is an antibody (Ab) which binds HER2. That is, the conjugates described herein are conjugates comprising antibodies which specifically bind to HER2.
  • telomere binding refers to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen).
  • the antibody or other molecule binds with an affinity of at least about 1 x107 M-1 , and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
  • a non-specific molecule e.g., BSA, casein
  • HER2 refers to Human Epidermal Growth Factor Receptor 2.
  • HER2 polypeptide corresponds to Genbank accession no. AAA75493, version no. AAA75493.1 Gl:306840, record update date: Jun 23, 2010 08:47 AM.
  • the nucleic acid encoding HER2 polypeptide corresponds to Genbank accession no. M1 1730, version no. M1 1730.1 Gl:183986, record update date: Jun 23, 2010 08:47 AM.
  • the antibody is an antibody that binds to HER2, 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 HER2.
  • 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 competes with the antibody secreted by hybridoma ATCC accession No. CRL-10463 for binding to HER2. In one embodiment the antibody binds HER2 with an association constant (Ka) no less than 2, 5 or 10-fold less than the antibody secreted by the hybridoma.
  • Ka association constant
  • the antibody is the antibody secreted by a hydridoma.
  • the hybridoma is ATCC accession No. CRL-10463.
  • 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.
  • 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.
  • the antigen binding portion for example minibodies
  • anti-idiotypic (anti-Id) antibodies intrabodies, and epitope-binding fragments of any of the above, so long as they exhibit the desired biological activity, for example, the ability to bind the cognate antigen.
  • 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.
  • 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.
  • allotype e.g. human G1 ml , G1 m2, G1 m3, non-G1 m1 [that, is any allotype other than G1 m1], G1 m17, G2m23, G3
  • 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 C1 q 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 or a portion thereof
  • 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 lgG1 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.
  • 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
  • substitutions are those wherein one amino acid is substituted for another within the groups of amino acids indicated herein below:
  • Particular preferred conservative amino acids substitution groups are: Val-Leu-lle, 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. Humanisation
  • 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.
  • CDR grafting includes '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).
  • 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.
  • 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 VH or Vi_ domain of a given non-human antibody specific for a particular epitope with a human VH or Vi_ 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 VHxVL combination. The method is described in Nature Biotechnology (N.Y.) 12, (1994) 899-903.
  • Composite antibodies In this method, 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: (a) determining the conformational structure of the variable region of the non-human (e.g. rodent) antibody (or fragment thereof) by constructing a three-dimensional model of the non-human antibody variable region;
  • step (c) defining for the non-human antibody to be humanized, a set of heavy and light chain surface exposed amino acid residues using the set of framework positions generated in step (b);
  • step (d) identifying from human antibody amino acid sequences a set of heavy and light chain surface exposed amino acid residues that is most closely identical to the set of surface exposed amino acid residues defined in step (c), wherein the heavy and light chain from the human antibody are or are not naturally paired;
  • step (e) substituting, in the amino acid sequence of the non-human antibody to be humanized, the set of heavy and light chain surface exposed amino acid residues defined in step (c) with the set of heavy and light chain surface exposed amino acid residues identified in step (d);
  • step (f) constructing a three-dimensional model of the variable region of the non-human antibody resulting from the substituting specified in step (e);
  • step (h) changing any residues identified in step (g) from the human to the original non- human amino acid residue to thereby define a non-human antibody humanizing set of surface exposed amino acid residues; with the proviso that step (a) need not be conducted first, but must be conducted prior to step (g).
  • 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. Human String Content Optimization
  • 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
  • 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 VH domain and a Vi_ 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 Vi_ domains with amino acid sequences that are identical to the sequences described herein. Methods of Treatment
  • the compounds of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula I.
  • a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula I.
  • terapéuticaally effective amount is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
  • a conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
  • compositions according to the present invention may comprise, in addition to the active ingredient, i.e. a conjugate of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. a conjugate of formula I
  • Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
  • Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the Conjugates can be used to treat proliferative disease and autoimmune disease.
  • proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
  • Cancers of particular interest include, but are not limited to breast cancers, gastric cancers, bladder cancers, prostate cancers, leukemias and ovarian cancers.
  • autoimmune disease examples include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis,
  • autoimmune demyelinative diseases e.g
  • Hashimoto's thyroiditis autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus
  • erythematosus hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti
  • granulomatosis granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing
  • the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1 -lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocyt.es (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn
  • B lymphocytes e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoi
  • the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. Drug loading
  • the drug loading (p) is the average number of PBD drugs per cell binding agent, e.g.
  • drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1 , 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent.
  • Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8.
  • the average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
  • the quantitative distribution of ADC in terms of p may also be determined.
  • ELISA the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 1 1 :843-852).
  • the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA.
  • ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues.
  • separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • Such techniques are also applicable to other types of conjugates.
  • p may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • Higher drug loading, e.g. p >5 may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.
  • an antibody may contain, for example, many lysine residues that do not react with the Drug Linker (A ) . Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety.
  • ADC ADC
  • DTT dithiothreitol
  • TCEP TCEP
  • the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker (A or B) relative to antibody, (ii) limiting the conjugation reaction time or
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
  • a reducing agent such as DTT (dithiothreitol).
  • DTT dithiothreitol
  • Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
  • Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
  • Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 1 14(13):2721 -2729; US 7521541 ; US 7723485; WO2009/052249).
  • the engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties.
  • the location of the drug moiety can thus be designed, controlled, and known.
  • the drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield.
  • Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody.
  • a drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.
  • the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1 , 2, 3, etc.
  • Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value.
  • Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.
  • antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
  • the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.
  • the Drug Linker compound used in the present invention (A) may be synthesised according to the Examples.
  • Conjugates can be prepared as previously described.
  • Antibodies can be conjugated to the Drug Linker compound (A) as described in Doronina et al., Nature Biotechnology, 2003, 21 , 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37 °C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved.
  • TCEP tris(carboxyethyl)phosphine hydrochloride
  • the reduced antibody is then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 ⁇ syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC- quenched drug-linker.
  • the C1 1 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
  • the C1 1 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
  • An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID N0.1 10, 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;
  • each of the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 is substituted by an amino acid that is not cysteine;
  • 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 103 of SEQ ID N0.1 10.
  • the cysteines at positions 109 and 1 12 in SEQ ID NO: 1 10 are substituted by 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.
  • An antibody comprising:
  • a light chain comprising the amino acid sequence of SEQ ID NO.151 ,
  • VL domain having the sequence SEQ ID NO. 2.
  • An antibody comprising:
  • a light chain comprising the amino acid sequence of SEQ ID NO.151 ,
  • VL domain having the sequence SEQ ID NO. 2.
  • An antibody comprising:
  • a light chain comprising the amino acid sequence of SEQ ID NO.151 ;
  • VH domain having the sequence SEQ ID NO. 1 ;
  • VL domain having the sequence SEQ ID NO. 2.
  • An antibody comprising:
  • a light chain comprising the amino acid sequence of SEQ ID NO.152;
  • VH domain having the sequence SEQ ID NO. 1 ;
  • An antibody comprising:
  • a light chain comprising the amino acid sequence of SEQ ID NO.153;
  • VH domain having the sequence SEQ ID NO. 1 ;
  • VL domain having the sequence SEQ ID NO. 2.
  • TLC thin-layer chromatography
  • Merck Kieselgel 60 F254 silica gel with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. Extraction and chromatography solvents were bought and used without further purification from VWR, U.K. All chemicals were purchased from Aldrich.
  • Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a Bruker AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz. Column chromatography was performed on an Isolera (Biotage) automated system using normal phase SNAP cartridges.
  • LCMS data were obtained using a Shimadzu Nexera series LC/MS with a Shimadzu LCMS-2020 quadrupole MS, with Electrospray ionisation.
  • Mobile phase A - 0.1 % formic acid in water.
  • Mobile phase B - 0.1 % formic acid in acetonitrile.
  • Short run gradient initial composition was 5% B held over 0.25 min, then increase from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutes and hold there for 0.05 min. Total gradient run time equals 3 min. Flow rate 0.8 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50°C. Column: Waters Acq uity UPLC BEH Shield RP18 1.7 ⁇ 2.1 x50mm.
  • the organic layer was separated and the aqueous phase was extracted with ethylacetate.
  • the combined organic layers were washed with water brined and dried over magnesium sulphate.
  • the ethylacetate was removed by rotary evaporation under reduced pressure to give the crude product as a yellow oil.
  • the crude product was purified by flash chromatography [85% n-hexane/15% ethylacetate] to afford the product as a colourless oil (10.74 g, 93%).
  • Diisopropyl azodicarboxylate (71.3 mL, 73.2 g, 362 mmol) was added drop-wise over a period of 60 min to an overhead stirred solution of methyl vanillate 4 (60 g, 329 mmol) and Ph 3 P (129.4 g, 494 mmol) in anhydrous THF (800 mL) at 0-5 °C (ice/acetone) under a nitrogen atmosphere.
  • Solid Cu(NC>3)2.3H20 (81 .54 g, 337.5 mmol) was added slowly to an overhead stirred slurry of the b/s-ester 5 (54.68 g, 135 mmol) in acetic anhydride (650 mL) at 0-5 °C (ice/acetone). The reaction mixture was allowed to stir for 1 h at 0-5 °C and then allowed to warm to room temperature. A mild exotherm (c. 40-50 °C), accompanied by thickening of the mixture and evolution of NO2 was observed at this stage. Additional acetic anhydride (300 mL) was added and the reaction mixture was allowed to stir for 16 h at room temperature.
  • the solid product was collected by vacuum filtration, washed with additional ether and dried in vacuo at 40 °C for 1 .5 h. This solid was then added portion wise to a suspension of the C-ring 3 (15.1 g, 84.9 mmol, 2.2 eq.) and TEA (19.5 g, 27 ml, 1 19.6 mmol, 5 eq.) in dry DCM (375 mL), maintaining the temperature between -40 and -50 °C with the aid of a dry ice/acetonitrile bath. The reaction mixture was allowed to stir at -40 °C for 1 h and then allowed to warm to room temperature at which point LCMS indicated the complete consumption of the starting material.
  • reaction mixture was diluted with additional DCM and washed sequentially with aqueous hydrochloric acid (1 M, 2 x 200 mL), saturated aqueous sodium bicarbonate (2 x 250 mL), water (250 mL), brine (250 mL), dried (MgS0 4 ). DCM was removed by rotary evaporation under reduced pressure to afford the product as a yellow foam (25.72 g, 94 %).
  • Solid lithium borohydride (3.18 g, 146 mmol, 3 eq.) was added in one portion to a solution of the ester 8 (34.72 g, 48.7 mmol, 1 eq.) in dry THF (350 mL) under a nitrogen
  • the activated Zinc was added to a solution of the nitro-TBS compound 10 (10.8 g, 12.2 mmol, 1 eq.) in MeOH (88 mL) and 5% formic acid/MeOH solution (440 ml_). The temperature rose to 37°C and the reaction mixture changed from a yellow to a colourless solution. Once the exotherm had subsided (20 min.) the reaction was shown to be complete by LCMS. The reaction mixture was filtered through celite washing with EtOAc. The EtOAc portion was washed with saturated bicarbonate solution (x 4) [caution effervescence!], water (x 1 ), brine (x 1 ), dried (MgS04) and evaporated under reduced pressure to give a yellow solid.
  • EEDQ (4.79 g, 19.3 mmol, 1 .05 eq.) was added to a solution of p-aminobenzyl alcohol (2.38 g, 19.3 mmol, 1 .05 eq.) and Alloc-Val-Ala-OH 16 (5.02 g, 18.4 mmol, 1 .0eq) in dry THF (100 mL). The mixture was stirred at room temperature for 72h. The solvent was evaporated under reduced pressure to give a pale brown solid. The solid was triturated with diethyl ether and filtered washing with an excess of diethyl ether. This afforded the product as a white solid (6.2 g, 89%). Analytical Data: RT 2.50 min; MS (ES + ) m/z (relative intensity) 400.6 ([Af + Na] + , 50), 378.6 ⁇ [M + 1 ] + , 60).
  • Triethylamine (0.38 g, 0.53 mL, 3.8 mmol, 2.2 eq.) was added to a stirred solution of the mono-boc protected b/ ' s-aniline (12) (1 .6 g, 1 .72 mmol, 1.0 eq.) and triphosgene (0.184 g, 0.62 mmol, 0.36 eq.) in dry THF (25 mL) under a nitrogen atmosphere at room temperature. The reaction mixture was heated to 40°C, after 5 min a sample was treated with methanol and analysed by LCMS as the methyl carbamate. Analytical Data: RT 2.32 min; MS (ES + ) m/z (relative intensity) 983 ([Af + H] + , 55), 1005 ([Af + Na] + , 100)
  • EDCI .HCI (0.13 g, 0.66 mmol, 1 .1 eq.) was added to a cloudy solution of compound 21 (0.61 g, 0.6 mmol, 1 .0 eq.) and Mal-dPEG 8 ®-OH (0.393 g, 0.66 mmol, 1 .1 eq.) in CHCI 3 (25 mL).
  • CHCI 3 25 mL
  • the clear solution was stirred at room temperature for 1 .5h., diluted with CHC (100 mL) washed with brine (2 x 100 mL), dried (MgS0 4 ) and evaporated under reduced pressure to give a yellow foam.
  • Boc anhydride (21 .6 g, 31 .7 mmol) was added to a solution of the diamine (25) (6.92 g 31 .7 mmol) in THF (200 mL) at room temperature. The resulting solution was then heated at reflux for 3 hours, cooled and evaporated to dryness under reduced pressure. The resulting residue was purified by column chromatography (70-100% ethyl acetate / hexane) to give the product as a pale yellow solid.
  • Triethyl amine (0.57 g, 5.6 mmol) was added in one go to a solution of the amine (26) (2 g, 2.56 mmol) and triphosgene (0.27 g, 0.92 mmol) in THF (30 mL) under nitrogen. The resulting mixture was heated at 40°C for 5 min. A small aliquot was quenched with methanol, and LCMS indicated complete conversion to the methyl carbamate (m/z 983, M+1 ).
  • Triethylamine (1 .22 g, 1.7 mL, 12.1 mmol, 2.2 eq.) was added to a stirred solution of the mono-boc protected b/ ' s-aniline (28) (5.0 g, 5.5 mmol, 1.0 eq.) and triphosgene (0.59 g, 1 .98 mmol, 0.36 eq.) in dry THF (75 mL) under a nitrogen atmosphere at room
  • IBX (45 wt%, 2.06 g, 3.3 mmol, 2.4 eq.) was added to a solution of the b/ ' s-alcohol 30 (1 .49 g, 1 .38 mmol, 1 .0 eq.) in anhydrous DMSO (40 mL). The solution was stirred at 30°C for 18h. LCMS analysis indicated the presence of a small amount of partially cyclised material. A further portion of IBX (45 wt%, 0.171 g, 0.275 mmol, 0.2 eq.) was added and the reaction was continued for a further 24h.
  • Pd(PPh 3 )4 (44 mg, 38.5 ⁇ , 0.04 eq.) was added to a solution of the cyclised product 31 (1 .04 g, 0.96 mmol, 1.0 eq.) and pyrrolidine (0.171 mg, 196 ⁇ , 2.4 mmol, 2.5 eq.) in anhydrous DCM (30 mL). The solution was stirred at room temperature for 30 min. The reaction mixture was diluted with DCM (30 mL) and washed with saturated NH4CI (100 mL), saturated brine (100 mL), dried (MgS0 4 ) and evaporated to give an off white foam.
  • EDCI.HCI (0.203 g, 1 .06 mmol, 1 .1 eq.) was added to a solution of compound 32 (0.86 g, 0.96 mmol, 1.0 eq.) and Mal-dPEG 8 ®-OH (0.57 g, 0.96 mmol, 1 .1 eq.) in dry DCM (30 mL) and CHC (to give a clear solution). The clear solution was stirred at room temperature for 18h. then a further portion of EDCI.HCI (0.037 g, 0.19 mmol, 0.2 eq.) was added and reaction continued for a further 24h.
  • Antibody and antibody-drug conjugate was assessed for monomeric content using size exclusion chromatography.
  • Running conditions Agilent 1 100 HPLC, TOSOH TSKgel G3000SWXL 7.8mm x 30cm, 5 ⁇ column, 0.5mL/min in, 0.2M Potassium Phosphate, 0.25M Potassium Chloride, 10% I PA, pH 6.95.
  • HIC Drug loading of the antibody was assessed using a combination of HIC and reverse phase chromatography.
  • HIC was carried out using a TOSOH Butyl-NPR 4.6mm x 3.5cm, 2.5 ⁇ column run at 0.8mL/min with a 12 minute linear gradient between A - 1 .5M (NH 4 )2S0 4 , 25mM NaPi, pH 6.95 ⁇ 0.05 and B - 75% 25mM NaPi, pH 6.95 ⁇ 0.05, 25% IPA.
  • Residual DMA solvent was determined by reverse phase HPLC using an Agilent Eclipse plus C18, 3.5 ⁇ , 4.6 x 100 mm Column at 35 ° C with a mobile phase of 5% CH 3 CN/95% 10mM Phosphate, 140mM NaCI, pH 6.5 ⁇ 0.1. Flow rate of 1 ml/min over 10 mins. A series of DMA standards in 30 mM Histidine, 200mM Sorbitol pH 6 ranging from 0.001 - 0.05 % v/v were analysed and a calibration curve generated. The level of DMA in the conjugates was determined with neat 10 ⁇ _ sample injections.
  • the antibody used in the following examples has a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2.
  • the constant domain comprises SEQ ID NO: 1 14 and 151 .
  • TCEP tris(2-carboxyethyl)phosphine
  • the buffer exchanged/reduced antibody was pooled (54mL at 3.76mg/mL) and conjugated by the addition of 3 equivalents of Compound 23 from a 10 mM stock in DMA (additional DMA was added prior to the Compound 23 addition to achieve 5% v/v final DMA during conjugation).
  • the conjugation reaction was mixed slowly for 60 minutes at 20°C before the reaction was stopped by adding 3 equivalents on NAC from a 10 mM stock in water and incubating for a further 30 minutes at 20°C.
  • the conjugated antibody was then
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • PBS phosphate- buffered saline pH 7.4
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • PBS phosphate- buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation, into a conjugation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent, and sterile-filtered.
  • Compound 23 was added as a DMSO solution (10 molar equivalent/antibody, 1 .38 micromoles, in 2.1 mL DMSO) to 18.5 mL of this reduced antibody solution (20.75 mg, 138 nanomoles) for a 10% (v/v) final DMSO concentration at a final antibody concentration of 1 .0 mg/mL.
  • the solution was mixed for ⁇ 1 .5 hours at room temperature, then the conjugation was quenched by addition of /V-acetyl cysteine (6 micromoles, 60 ⁇ - at 100 mM), and the conjugate was purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.
  • mice 8 to 12 weeks old CB.17 SCI D mice were implanted with 1 x10 7 tumor cells in 50% Matrigel s.c. in the flank.
  • Figure 1 shows the effect of tumour volume of the vehicle (O), B12-23 dosed at 1 mg/kg (O), or Antibody-23 dosed at 1 mg/kg ( ⁇ ).
  • Figure 2 shows the effect of tumour volume of the vehicle (O), B12-23 dosed at 3 mg/kg (V), or Antibody-23 dosed at 3 mg/kg ( ⁇ ).
  • mice 5 weeks old athymic nude - Foxn1 nu mice weighing at least 18 grams were allocated to acclimate in the animal facility with access to food and water ad libitum for at least 6 days prior to manipulation. Tumors of the same passage were transplanted subcutaneously onto 5-10 mice (donor mice, passage (n-1 )). When these tumors reached 1000 to 2000 mm 3 , donor mice were sacrificed by cervical dislocation, tumors were aseptically excised and dissected. After removing necrotic areas, tumors were cut into fragments measuring approximately 20 mm 3 and transferred in culture medium before grafting.
  • Figure 3 shows the effect of tumour volume of the vehicle (O), B12-23 dosed at 1 mg/kg (V),B12-23 dosed at 3 mg/kg (O - upper line), Antibody-23 dosed at 1 mg/kg ( ⁇ ),
  • Antibody-23 dosed at 3 mg/kg ( - lower line).
  • the therapeutic index (Tl) of a drug-linker in conjugates may be calculated using the maximum tolerated dose (MTD) divided by the minimum effective dose (MED).
  • MTD maximum tolerated dose
  • MED minimum effective dose
  • the MTD and MED may be body surface area adjusted (according to the method in Reagan-Shaw, S., et al., The FASEB Journal, 22, 659-662, 2007; doi: 10.1096/fj.07-9574LSF).
  • the MED is defined as the dose resulting in at least 21 days tumour stasis in a mouse pharmacology model, with the MTD defined as a dose resulting in non-lethal / non-life threatening toxicity in a rat.
  • the MTD in rate of the non-binding conjugate of 23 of > 8 mg/kg has a HED of > 1.3 mg/kg (> 8 mg/kg * 6/37).
  • Tl is > 16.
  • mice 5 weeks old athymic nude - Foxn1 nu mice weighing at least 18 grams were allocated to acclimate in the animal facility with access to food and water ad libitum for at least 6 days prior to manipulation. Tumors of the same passage were transplanted subcutaneously onto 6-24 mice (donor mice, passage (n-1 )). When these tumors reached 1000 to 2000 mm 3 , donor mice were sacrificed by cervical dislocation, tumors were aseptically excised and dissected. After removing necrotic areas, tumors were cut into fragments measuring approximately 20 mm 3 and transferred in culture medium before grafting.
  • a second set of athymic nude - Foxn1 nu mice were anaesthetized with 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine, and then skin was aseptized with a chlorhexidine solution, incised at the level of the interscapular region, and a 20 mm 3 tumor fragment was placed in the subcutaneous tissue. Skin was closed with clips. All mice from the same experiment were implanted on the same day.
  • Figure 4 shows the effect of tumour volume of the vehicle (O), B12-23 dosed at 0.5 mg/kg (V), Antibody-23 dosed at 0.25 mg/kg (O) and Antibody-23 dosed at 0.5 mg/kg (V).
  • SEQ ID NO. 1 (Herceptin VH):
  • SEQ ID NO. 1 10 (lgG1 HC constant region)
  • SEQ ID NO. 1 14 (lgG1 HC constant region, BJ C ⁇ V)
  • SEQ ID NO. 120 (lgG2 HC constant region) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSPGK
  • SEQ ID NO. 130 (lgG3 HC constant region)
  • SEQ ID NO. 140 (lgG4 HC constant region)
  • SEQ ID NO. 162 (ALC constant region, C102V)
  • SEQ ID NO. 163 (ALC constant region, C102&S103del)

Landscapes

  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un conjugué de formule L- (DL)p (I), dans laquelle L est un anticorps (Ab) qui se lie à HER2, D représente une unité lieur de médicament de formule (A) dans laquelle p est un entier de 1 à 20.
PCT/EP2017/052991 2016-02-10 2017-02-10 Conjugués anticorps anti-her2-pyrrolobenzodiazépine Ceased WO2017137556A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1602363.2 2016-02-10
GBGB1602363.2A GB201602363D0 (en) 2016-02-10 2016-02-10 Pyrrolobenzodiazepine conjugates

Publications (1)

Publication Number Publication Date
WO2017137556A1 true WO2017137556A1 (fr) 2017-08-17

Family

ID=55642076

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/052991 Ceased WO2017137556A1 (fr) 2016-02-10 2017-02-10 Conjugués anticorps anti-her2-pyrrolobenzodiazépine

Country Status (2)

Country Link
GB (1) GB201602363D0 (fr)
WO (1) WO2017137556A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019065964A1 (fr) * 2017-09-29 2019-04-04 第一三共株式会社 Conjugué anticorps-dérivé de pyrrolobenzodiazépine
WO2020196712A1 (fr) 2019-03-27 2020-10-01 第一三共株式会社 Combinaison d'un conjugué anticorps-dérivé de pyrrolobenzodiazépine et d'un inhibiteur de parp
JPWO2020196475A1 (fr) * 2019-03-25 2020-10-01
WO2020196474A1 (fr) 2019-03-25 2020-10-01 第一三共株式会社 Conjugué anticorps-dérivé de pyrrolobenzodiazépine
CN113166764A (zh) * 2018-11-14 2021-07-23 第一三共株式会社 抗cdh6抗体-吡咯并苯并二氮杂环庚三烯衍生物缀合物
US11484606B2 (en) 2019-06-07 2022-11-01 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2024005123A1 (fr) 2022-06-30 2024-01-04 東レ株式会社 Composition pharmaceutique pour le traitement et/ou la prévention du cancer
US11976122B2 (en) 2020-07-31 2024-05-07 Adc Therapeutics Sa Anti-IL13Rα2 antibodies
RU2820928C2 (ru) * 2017-09-29 2024-06-13 Даити Санкио Компани, Лимитед Конъюгат антитело-производное пирролобензодиазепина

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006065533A2 (fr) * 2004-11-29 2006-06-22 Seattle Genetics, Inc. Anticorps et immunoconjugues mis au point
WO2011130598A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines et conjugués de celles-ci
WO2015031693A1 (fr) * 2013-08-28 2015-03-05 Stem Centrx, Inc. Conjugués anti-dll3 modifiés et procédés d'utilisation
WO2016166300A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués anticorps-médicament spécifiques à un site

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006065533A2 (fr) * 2004-11-29 2006-06-22 Seattle Genetics, Inc. Anticorps et immunoconjugues mis au point
WO2011130598A1 (fr) * 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazépines et conjugués de celles-ci
WO2015031693A1 (fr) * 2013-08-28 2015-03-05 Stem Centrx, Inc. Conjugués anti-dll3 modifiés et procédés d'utilisation
WO2016166300A1 (fr) * 2015-04-15 2016-10-20 Van Berkel Patricius Hendrikus Cornelis Conjugués anticorps-médicament spécifiques à un site

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALLEY M C ET AL: "SJG-136 (NSC 694501), A NOVEL RATIONALLY DESIGNED DNA MINOR GROOVE INTERSTRAND CROSS-LINKING AGENT WITH POTENT AND BROAD SPECTRUM ANTITUMOR ACTIVITY. PART 2: EFFICACY EVALUATIONS", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 64, no. 18, 1 January 2004 (2004-01-01), pages 6700 - 6706, XP008055764, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-03-2942 *
HONGCHENG LIU ET AL: "Disulfide bond structures of IgG molecules: Structural variations, chemical modifications and possible impacts to stability and biological function", MABS, vol. 4, no. 1, 1 January 2012 (2012-01-01), pages 17 - 23, XP055134217, ISSN: 1942-0862, DOI: 10.4161/mabs.4.1.18347 *

Cited By (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI820044B (zh) * 2017-09-29 2023-11-01 日商第一三共股份有限公司 抗體－吡咯并苯二氮呯衍生物複合體
JP2023062095A (ja) * 2017-09-29 2023-05-02 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
JPWO2019065964A1 (ja) * 2017-09-29 2020-09-10 第一三共株式会社 抗体−ピロロベンゾジアゼピン誘導体コンジュゲート
EP3690038A4 (fr) * 2017-09-29 2021-05-19 Daiichi Sankyo Company, Limited Conjugué anticorps-dérivé de pyrrolobenzodiazépine
TWI885539B (zh) * 2017-09-29 2025-06-01 日商第一三共股份有限公司 抗體－吡咯并苯二氮呯衍生物複合體
US12246196B2 (en) 2017-09-29 2025-03-11 Daiichi Sankyo Company, Limited Antibody-drug conjugates comprising substituted benzo[e]pyrrolo[1,2-a][1,4]diazepines
JP7556073B2 (ja) 2017-09-29 2024-09-25 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
RU2820928C2 (ru) * 2017-09-29 2024-06-13 Даити Санкио Компани, Лимитед Конъюгат антитело-производное пирролобензодиазепина
CN111164208A (zh) * 2017-09-29 2020-05-15 第一三共株式会社 抗体－吡咯并苯并二氮杂卓衍生物偶联物
US11583590B2 (en) 2017-09-29 2023-02-21 Daiichi Sankyo Company, Limited Antibody-pyrrolobenzodiazepine derivative conjugate and method of use thereof for treating a tumor
US12350344B2 (en) 2017-09-29 2025-07-08 Daiichi Sankyo Company, Limited Methods of treating a tumor by administering a claudin-6 (CLDN6) or CLDN9 antibody-pyrrolobenzodiazepine derivative conjugate
CN111164208B (zh) * 2017-09-29 2023-08-04 第一三共株式会社 抗体－吡咯并苯并二氮杂卓衍生物偶联物
JP7273716B2 (ja) 2017-09-29 2023-05-15 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
WO2019065964A1 (fr) * 2017-09-29 2019-04-04 第一三共株式会社 Conjugué anticorps-dérivé de pyrrolobenzodiazépine
US11628223B2 (en) 2017-09-29 2023-04-18 Daiichi Sankyo Company, Limited Antibody-drug conjugates comprising substituted benzo[e]pyrrolo[1,2-α][1,4]diazepines
JP7133079B2 (ja) 2017-09-29 2022-09-07 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
JP2022036943A (ja) * 2017-09-29 2022-03-08 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
EP3882349A4 (fr) * 2018-11-14 2023-04-05 Daiichi Sankyo Company, Limited Conjugué (anticorps anti-cdh6)-(dérivé de pyrrolobenzodiazépine)
CN113166764A (zh) * 2018-11-14 2021-07-23 第一三共株式会社 抗cdh6抗体-吡咯并苯并二氮杂环庚三烯衍生物缀合物
EP3950061A4 (fr) * 2019-03-25 2022-11-16 Daiichi Sankyo Company, Limited Conjugué anticorps-dérivé de pyrrolobenzodiazépine
JPWO2020196474A1 (fr) * 2019-03-25 2020-10-01
CN113631229B (zh) * 2019-03-25 2025-09-23 第一三共株式会社 抗体－吡咯并苯并二氮杂卓衍生物偶联物
EP3949987A4 (fr) * 2019-03-25 2023-02-22 Daiichi Sankyo Company, Limited Conjugué anticorps anti-her2/dérivé de pyrrolobenzodiazépine
JPWO2020196475A1 (fr) * 2019-03-25 2020-10-01
KR20210143237A (ko) 2019-03-25 2021-11-26 다이이찌 산쿄 가부시키가이샤 항체-피롤로벤조디아제핀 유도체 컨쥬게이트
KR20210141630A (ko) 2019-03-25 2021-11-23 다이이찌 산쿄 가부시키가이샤 항 her2 항체-피롤로벤조디아제핀 유도체 콘주게이트
CN113631229A (zh) * 2019-03-25 2021-11-09 第一三共株式会社 抗体－吡咯并苯并二氮杂卓衍生物偶联物
WO2020196474A1 (fr) 2019-03-25 2020-10-01 第一三共株式会社 Conjugué anticorps-dérivé de pyrrolobenzodiazépine
WO2020196475A1 (fr) 2019-03-25 2020-10-01 第一三共株式会社 Conjugué anticorps anti-her2/dérivé de pyrrolobenzodiazépine
JP7627655B2 (ja) 2019-03-25 2025-02-06 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
TWI856078B (zh) * 2019-03-25 2024-09-21 日商第一三共股份有限公司 抗體–吡咯并苯二氮呯衍生物結合物、其製造方法及其用途
JP7529654B2 (ja) 2019-03-25 2024-08-06 第一三共株式会社 抗ｈｅｒ２抗体－ピロロベンゾジアゼピン誘導体コンジュゲート
JP7522097B2 (ja) 2019-03-27 2024-07-24 第一三共株式会社 抗体－ピロロベンゾジアゼピン誘導体コンジュゲートとｐａｒｐ阻害剤の組み合わせ
JPWO2020196712A1 (fr) * 2019-03-27 2020-10-01
EP3949988A4 (fr) * 2019-03-27 2022-11-16 Daiichi Sankyo Company, Limited Combinaison d'un conjugué anticorps-dérivé de pyrrolobenzodiazépine et d'un inhibiteur de parp
CN113631191A (zh) * 2019-03-27 2021-11-09 第一三共株式会社 抗体－吡咯并苯并二氮杂卓衍生物偶联物与parp抑制剂的组合
KR20210143839A (ko) 2019-03-27 2021-11-29 다이이찌 산쿄 가부시키가이샤 항체-피롤로벤조디아제핀 유도체 콘주게이트와 parp 저해제의 조합
WO2020196712A1 (fr) 2019-03-27 2020-10-01 第一三共株式会社 Combinaison d'un conjugué anticorps-dérivé de pyrrolobenzodiazépine et d'un inhibiteur de parp
US11484606B2 (en) 2019-06-07 2022-11-01 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11976122B2 (en) 2020-07-31 2024-05-07 Adc Therapeutics Sa Anti-IL13Rα2 antibodies
WO2024005123A1 (fr) 2022-06-30 2024-01-04 東レ株式会社 Composition pharmaceutique pour le traitement et/ou la prévention du cancer

Also Published As

Publication number Publication date
GB201602363D0 (en) 2016-03-23

Similar Documents

Publication Publication Date Title
JP7523506B2 (ja) 抗体－薬物コンジュゲート
JP7132311B2 (ja) 細胞障害性ベンゾジアゼピン誘導体
US20230143309A1 (en) Pyrrolobenzodiazepine antibody conjugates
WO2017137556A1 (fr) Conjugués anticorps anti-her2-pyrrolobenzodiazépine
ES2731681T3 (es) Conjugados de anticuerpo anti-DLL3 y PBD y usos de los mismos
ES2982012T3 (es) Materiales biológicos y usos de los mismos
ES2670937T3 (es) Conjugados de anticuerpo-pirrolobenzodiazepina
US20190209704A1 (en) Novel antibody-drug conjugates and related compounds, compositions and methods of use
CN103288957B (zh) 一种抑制肿瘤生长的抗体药物衍生物及其制备方法和用途
WO2015155345A1 (fr) Anticorps et conjugués anticorps-médicament
WO2015038426A1 (fr) Lieurs auto-immolables contenant des dérivés d'acide mandélique, conjugués médicament-ligand pour thérapies ciblées, et leurs utilisations
CN105102455A (zh) 亲水性自消耗连接子及其缀合物
JP2024512986A (ja) 改良された物理化学的特性および薬理学的特性を有する酵素トリガー自己反応性リンカー
ES2690067T9 (es) Conjugados de anticuerpos anti-PSMA-Pirrolobenzodiazepinas
CN106661124A (zh) 抗叶酸受体α(FRA)抗体‑药物缀合物及其使用方法
JP2020531471A (ja) ピロロベンゾジアゼピン複合体
WO2020025108A1 (fr) Lieurs et conjugués
HK40002093B (en) Pyrrolobenzodiazepine conjugates
HK40002093A (en) Pyrrolobenzodiazepine conjugates
HK1232895B (zh) 抗c-met抗体和抗c-met抗体-细胞毒性药物偶联物及其医药用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17704742

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17704742

Country of ref document: EP

Kind code of ref document: A1