WO2025111431A1 - Conjugués anticorps-médicament anti-cd7 et leurs procédés d'utilisation - Google Patents

Conjugués anticorps-médicament anti-cd7 et leurs procédés d'utilisation Download PDF

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WO2025111431A1
WO2025111431A1 PCT/US2024/056834 US2024056834W WO2025111431A1 WO 2025111431 A1 WO2025111431 A1 WO 2025111431A1 US 2024056834 W US2024056834 W US 2024056834W WO 2025111431 A1 WO2025111431 A1 WO 2025111431A1
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group
antibody
alkyl
branched
linear
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Inventor
Matthew T. Burger
Zhuoliang Chen
Joseph Anthony D'ALESSIO
Eric Andrew Mcneill
Katsumasa Nakajima
Richard Vaughan Newcombe
Bing Yu
Qiang Zhang
Gregory John Hollingworth
Jean-baptiste LANGLOIS
Ana Leticia MARAGNO
Jerome-Benoit Starck
Laura BRESSON
Gaetane Le Toumelin-Braizat
Imre Fejes
Zoltan Szlavik
Stuart Ray
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Novartis AG
Les Laboratoires Servier SAS
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Novartis AG
Les Laboratoires Servier SAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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 disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody or an antigen-binding fragment thereof covalently linked to two antineoplastic compounds through a dual linker, wherein at least one antineoplastic payload is a BH3 mimetic.
  • ADCs antibody-drug conjugates
  • the disclosure further relates to methods and compositions useful in the treatment and/or diagnosis of cancers that express a target antigen (e.g. CD7) and/or are amenable to treatment by modulating expression and/or activity of Bcl-2 family proteins, as well as methods of making those compositions.
  • a target antigen e.g. CD7
  • Linker-drug conjugates comprising the dual linker and antineoplastic compounds (e.g., two BH3 mimetics or a BH3 mimetic moiety and an antineoplastic non-BH3 mimetic) and methods of making same are also disclosed.
  • antineoplastic compounds e.g., two BH3 mimetics or a BH3 mimetic moiety and an antineoplastic non-BH3 mimetic
  • Apoptosis (programmed cell death) is an evolutionarily conserved pathway essential for tissue homeostasis, development and removal of damaged cells. Deregulation of apoptosis contributes to human diseases, including malignancies, neurodegenerative disorders, diseases of the immune system and autoimmune diseases (Hanahan and Weinberg, Cell. 2011 Mar 4;144(5):646-74; Marsden and Strasser, Amu Rev Immunol. 2003;21:71-105; Vaux and Flavell, Curr Opin
  • the Bcl-2 protein family comprises key regulators of cell survival which can suppress (e.g., Bcl-2, Bcl-xL, Mcl-1) or promote (e.g, Bad, Bax) apoptosis (Gross et al., Genes Dev. 1999 Aug 1 ; 13(15): 1899-911, Youle and Strasser, Nat. Rev. Mol. Cell Biol. 2008 Jan;9(l):47-59).
  • the prosurvival members such as Bcl-2, Bcl-xL, and Mcl-1 contain BH domains 1-4, whereas Bax and Bak, the proapoptotic effectors of mitochondrial outer membrane permeabilization during apoptosis, contain BH domains 1-3 (Youle and Strasser, Nat. Rev. Mol. Cell Biol. 2008 Jan;9(l):47-59).
  • Bcl- xL also named BCL2L1, from BCL2-like 1
  • BCL2L1 from BCL2-like 1
  • T-cells isolated from the joints of rheumatoid arthritis patients exhibited increased Bcl-xL expression and were resistant to spontaneous apoptosis (Salmon et al., J Clin Invest. 1997 Feb 1 ;99(3) :439-46. doi: 10.1172/JCI119178.PMID: 9022077).
  • BH3 mimetics a new class of drugs named BH3 mimetics. These molecules are able to disrupt the interaction between the proapoptotic and antiapoptotic members of the Bcl-2 family and are potent inducers of apoptosis.
  • This new class of drugs includes inhibitors of Bcl-2, Bcl-xL, Bcl-w and Mcl-1.
  • the first BH3 mimetics described were ABT- 737 and ABT-263, targeting Bcl-2, Bcl-xL and Bcl-w (Park et al., J. Med. Chem. 2008 Nov 13;51(21):6902-15; Roberts et al., J. Clin. Oncol.
  • the selective Bcl-2 inhibitor ABT- 199 is now approved for the treatment of patients with CLL and AML in combination therapy, while the other inhibitors are still under pre-clinical or clinical development.
  • ABT-263 has shown activity in several hematological malignancies and solid tumors (Shoemaker etal., Clin. Cancer Res. 2008 Jun 1;14(11):3268-77; Ackler et al., Cancer Chemother. Pharmacol. 2010 Oct;66(5):869-80; Chen et al., Mol. Cancer Ther. 2011 Dec;10(12):2340-9).
  • ABT-263 exhibited objective antitumor activity in lymphoid malignancies (Wilson et al., Lancet Oncol. 2010 Dec; 11(12): 1149-59; Roberts et al., J. Clin. Oncol. 2012 Feb 10;30(5):488-96) and its activity is being investigated in combination with several therapies in solid tumors.
  • the selective Bcl-xL inhibitors, A-l 155463 or A-1331852 exhibited in vivo activity in pre-clinical models of T-ALL (T-cell Acute Lymphoblastic Leukemia) and different types of solid tumors (Tao et al., ACS Med. Chem. Lett.
  • ABT-737 prevented allogeneic T-cell activation, proliferation, and cytotoxicity in vitro and inhibited allogeneic T- and B- cell responses after skin transplantation with high selectivity for lymphoid cells (Cippa et al., .Transpl Int. 2011 Jul;24(7):722-32. doi: 10.1111/j.1432-2277.2011.01272.x. Epub 201 1 May 25.PMID: 21615547).
  • Bcl-2 family proteins e.g, Bcl-2, Bcl-xL, Mcl-1
  • upstream and/or downstream proteins in an apoptotic signaling pathway in oncology and in the field of immune and autoimmune diseases.
  • CD7 is a transmembrane glycoprotein expressed in normal T cells from early until late stages of maturation (Reinhold U et al. Immunology. 1996 Nov;89(3):391-6. doi: 10.1046/j.l365- 2567.1996. d01-744.x.PMID: 8958052).
  • Previous data have shown that CD7 may play a role in T-cell and T-cell/B-cell interactions during lymphoid development and has a co-stimulatory role with CD3, CD45 and PI3K (Chan AS et al, J Immunol. 1997 Jul 15;159(2):934-42.PMID: 9218614; Stillwell R and Bierer BE. Immunol Res. 2001 ;24(1):31 -52.
  • CD7 is highly expressed on almost all T-ALL patient samples (Pais H, et al, Sci Rep. 2019 Apr 8;9(l):5760. doi: 10.1038/s41598-019-42214-w.PMID: 30962539). CD7 is also expressed in 30% of AML cells and in a fewer proportion of other hematological cell malignancies (Del Poeta G et al, Leuk Lymphoma.
  • Anti-CD7 antibody based therapies have been generated in the past for the treatment of CD7 positive malignancies (Baum W, et al, Br J Haematol. 1996 Nov;95(2):327-38. doi: 10.1046/j.1365-2141.1996.d01-1900.x.PMID: 8904888; Peipp M et al, Cancer Res. 2002 May 15;62(10):2848-55.PMID: 1201916; Tang J et al, Oncotarget. 2016 Jun 7;7(23):34070- 83. doi: 10.18632/oncotarget.8710. PMID: 2708300; Frankel AE, et al, Leuk Lymphoma.
  • the present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof covalently linked to two antineoplastic payloads through a dual linker, wherein at least one antineoplastic payloads is a BH3 mimetic, and wherein the dual linker has one attachment point connected to the antibody and two attachment points to the two antineoplastic payloads, and wherein the two antineoplastic payloads can be the same or different, and wherein the antibody or antigen-binding fragment thereof binds to target antigen CD7; wherein the antibody or antigen-binding fragment thereof is not Ab D.
  • one antineoplastic payload is a BH3 mimetic and the other antineoplastic payload is an antineoplastic non-BH3 mimetic.
  • the antineoplastic non-BH3 mimetic is a topoisomerase 1 inhibitor or an antimitotic drug.
  • the topoisomerase 1 inhibitor is selected from topotecan, exatecan, deruxtecan and SN-38.
  • the anti-mitotic drug is monomethyl auristatin E (MMAE) or a taxane.
  • the taxane is selected from docetaxel, paclitaxel, or cabazitaxel.
  • said two antineoplastic payloads are two BH3 mimetics.
  • the BH3 mimetic is selected from a Mcl-1 inhibitor, a Bcl-2 inhibitor, and a Bcl-xL inhibitor.
  • the BH3 mimetic of said two antineoplastic payloads are the same. In some embodimetns, the BH3 mimetic of said two antineoplastic payloads are different.
  • the antineoplastic payloads in the antibody-drug conjugate of the present disclosure are defined as: (i) one antineoplastic payload is a Mcl-1 inhibitor and the other antineoplastic payload is a Bcl-2 inhibitor; (ii) one antineoplastic payload is a Mcl-1 inhibitor and the other antineoplastic payload is a Bcl-xL inhibitor; or (iii) one antineoplastic payload is a Bcl-2 inhibitor and the other antineoplastic payload is a Bcl-xL inhibitor.
  • one antineoplastic payload is a Mcl-1 inhibitor, a Bcl-2 inhibitor, and a Bcl-xL inhibitor
  • the other antineoplastic payload is a topoisomerase 1 inhibitor or an anti-mitotic drug.
  • one antineoplastic payload is a Bcl-xL inhibitor and the other antineoplastic payload is a topoisomerase 1 inhibitor.
  • one antineoplastic payload is a Bcl-xL inhibitor and the other antineoplastic payload is an anti-mitotic drug.
  • one antineoplastic payload is a Mcl- 1 inhibitor and the other antineoplastic payload is a topoisomerase 1 inhibitor.
  • one antineoplastic payload is a Mcl- 1 inhibitor and the other antineoplastic payload is an anti-mitotic drug.
  • one antineoplastic payload is a Bcl-2 inhibitor and the other antineoplastic payload is a topoisomerase 1 inhibitor.
  • one antineoplastic payload is a Bcl-2 inhibitor and the other antineoplastic payload is an anti-mitotic drug.
  • the present disclosure provides antibody-drug conjugate of the first embodiment, wherein the antibody-drug conjugate is represented by Eormula (A): wherein:
  • Ab is an anti-CD7 antibody or an antigen-binding fragment thereof
  • R 1 is an attachment group
  • L 1 is a bridging spacer
  • W is branching moiety
  • L 2 and L 3 ’ are each independently a linker
  • D 1 and D 2 are each independently an antineoplastic compound, wherein at least one of D 1 and D 2 is a BH3 mimetic; and a is an integer from 1 to 16. In some embodiments, D 1 and D 2 are each independently a BH3 mimetic.
  • the present disclosure provides an antibody-drug conjugate of the second embodiment, wherein a is an integer from 1 to 8, 1 to 6, 1 to 4, or a is 1 or 2, optionally wherein a is determined by liquid chromatography-mass spectrometry (LC-MS).
  • LC-MS liquid chromatography-mass spectrometry
  • the definitions of the remaining variables are provided in the second embodiment or any embodiments described therein.
  • a is an integer from 1 to 6 or from 1 to 4 or a is 1 or 2 or a is determined by liquid chromatography-mass spectrometry (LC-MS).
  • each of L 2 and L 3 comprises a cleavable group, optionally wherein at least one cleavable group comprises a glucuronide group, pyrophosphate group, a peptide group, and/or a self-immolative group.
  • each of L 2 and L 3 comprises a cleavable group, optionally at least one cleavable group comprises a pyrophosphate group, a peptide group and/or a self-immolative group.
  • the definitions of the remaining variables are provided in the second or third embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the second embodiment, wherein the antibody-drug conjugate is represented by Formula (B): wherein:
  • Ab is an anti-CD7 antibody or an antigen-binding fragment thereof
  • R 1 is an attachment group
  • L 1 is a bridging spacer
  • W is N or CR W ; wherein R w is H or Ci-ealkyl;
  • L 2 and L 3 are each independently a connecting spacer
  • E 1 and E 2 are each independently, an enzyme cleavage element or a hydrophilic moiety; V 1 and V 2 are each independently comprise: i) a self immolative group, ii) an enzyme cleavage element, or iii) a self-immolative group and an enzyme cleavage element; and D 1 and D 2 are each independently an antineoplastic compound, wherein at least one of D 1 and D 2 is a BH3 mimetic.
  • V 1 and V 2 are each independently i) a self immolative group or ii) an enzyme cleavage element; and D 1 and D 2 are each independently a BH3 mimetic.
  • the present disclosure provides an antibody-drug conjugate of the fifth embodiment, wherein the antibody-drug conjugate is represented by Formula (C): or pharmaceutically acceptable salt thereof, wherein
  • Ab is an anti-CD7 antibody or an antigen-binding fragment thereof
  • R 1 is an attachment group
  • L 1 is a bridging spacer
  • W is N or CR W ; wherein R w is H or Ci-ealkyl;
  • L 2 and L 3 are each independently a connecting spacer
  • E 1 and E 2 are each independently a peptide group comprising 1 to 6 amino acids, wherein said peptide group is optionally substituted by a hydrophilic group;
  • D 1 and D 2 are each independently an antineoplastic compound, wherein at least one of D 1 and D 2 is a BH3 mimetic;
  • L 4 and L 5 are each independently a spacer moiety
  • R 2 and R 3 are each independently a hydrophilic group or an enzyme cleavage element; and m and n are each independently 0 or 1.
  • D 1 and D 2 are each independently a BH3 mimetic.
  • the present disclosure provides an antibody-drug conjugate of the seventh embodiment, wherein the antibody-drug conjugate is represented by Formula (DI), (D2), or (D3):
  • D 1 and D 2 are each independently an antineoplastic compound, wherein at least one of D 1 and D 2 is a BH3 mimetic; R 2 and R 3 are each independently an enzyme cleavage element; and for Formula (D3), R 2 is a hydrophilic group and R 3 is an enzyme cleavage element.
  • D 1 and D 2 are each independently a BH3 mimetic.
  • the present disclosure provides an antibody-drug conjugate of the eighth embodiment, wherein for Formula (DI), R 2 and R 3 are each independently a hydrophilic group.
  • R 2 and R 3 are each independently a hydrophilic group.
  • the definitions of the remaining variables are provided in the eighth embodiment or any embodiment described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the ninth embodiments, wherein the attachment group is formed by a reaction comprising at least one reactive group.
  • the definitions of the remaining variables are provided in any one of the second through the ninth embodiments or any embodiment described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the tenth embodiments, wherein the attachment group is formed by reacting: a first reactive group that is attached to the linker, and a second reactive group that is attached to the antibody or is an amino acid residue of the antibody, wherein optionally,
  • At least one of the reactive groups comprises: a thiol, a maleimide, a haloacetamide, an azide, an alkyne, a cyclcooctene, a triaryl phosphine, an oxanobomadiene, a cyclooctyne, a diaryl tetrazine, a monoaryl tetrazine, a norbomene, an aldehyde, a hydroxylamine, a hydrazine,
  • each R 11 is independently selected from H and Ci-Cgalkyl
  • each R 12 is 2-pyridyl or 4-pyridyl
  • each R 13 is independently selected from H, Ci-Cgalkyl, F, Cl, and -OH
  • each R 14 is independently selected from H, Ci-Cgalkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, - N(CH 3 ) 2 , -CN, -NO 2 and -OH
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the eleventh embodiments, wherein the attachment group is selected from:
  • R 16 is H, CM alkyl, phenyl, pyrimidine or pyridine;
  • R 17 is independently selected from H, phenyl and pyridine; q is 0, 1, 2 or 3;
  • R 19 is H or methyl
  • R 20 is H, -CH3 or phenyl.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the twelfth embodiments, wherein the attachment group
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the thirteenth embodiments, wherein:
  • L 1 comprises:
  • n is an integer from 1 to 12, wherein the * of L 1 indicates the point of direct or indirect attachment to W, and the ** of L 1 indicates the point of direct or indirect attachment to R 1 ; integer from 1 to 12 or n is 1 or n is 12, wherein the * of L 1 indicates the point of direct or indirect attachment to W, and the ** of L 1 indicates the point of direct or indirect attachment to R 1 ;
  • L 1 is , and n is an integer from 1 to 12, wherein the * of L 1 indicates the point of direct or indirect attachment to W, and the ** of Li indicates the point of direct or indirect attachment to R 1 ;
  • L 1 comprises , wherein the * of L 1 indicates the point of direct or indirect attachment to W, and the ** of L 1 indicates the point of direct or indirect attachment to R 1 ;
  • L 1 is a bridging spacer comprising:
  • each R L1 is independently selected from H and Ci-Cgalkyl.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the fourteenth embodiments, wherein L 1 comprises a moiety represented by wherein n is an integer from 1 to 12, wherein the * of L 1 indicates the point of direct or indirect attachment to W, and the ** of L 1 indicates the point of direct or indirect attachment to R 1 .
  • the definitions of the remaining variables are provided in the second through the fourteenth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the fifteenth embodiment, wherein L 1 is represented by a formula wherein n is an integer from 1 to 12; x is an integer from 0 to 6; y is 0 or 1 ; z is an integer from 0 to 6; u is 0 or 1 ; and wherein the * of L 1 indicates the point of direct attachment to W, and the ** of L 1 indicates the point of direct attachment to R 1 .
  • the present disclosure provides an antibody-drug conjugate of any one of the second through the sixteenth embodiments, wherein L 1 is selected from the group consisting of :
  • the present disclosure provides an antibody-drug conjugate of the eighteenth embodiment, wherein L 2 and L 3 are each independently a connecting spacer selected from a group consisting of wherein k, in each occurrence, is independently an integer from 0 to 4; r, in each occurrence, is independently 0 or 1 ; o, in each occurrence, is independently an integer from 0 to 10; p, in each occurrence, is independently an integer from 0 to 4;
  • R L23 is hydrogen or Ci-ealkyl
  • R L is hydrogen or -C(O)-R H ;
  • R H is a hydrophilic group; and the # of L 2 or L 3 indicates the point of direct attachment to E 1 or E 2 , respectively, and the ## of L 2 or L 3 indicates the point of direct attachment to W; provided that when W is N, L 2 and L 3 are not (L2c), (L2d), (L2f), or (L2k).
  • the present disclosure provides an antibody-drug conjugate of the nineteenth embodiment, wherein L 2 and L 3 are each independently a connecting spacer selected from a group consisting of
  • R L23 is hydrogen or Cmalkyl
  • R L is hydrogen or -C(O)-R H ;
  • R H is a hydrophilic group; and the # of L 2 or L 3 indicates the point of direct attachment to E 1 or E 2 , respectively, and the ## of L 2 or L 3 indicates the point of direct attachment to W; provided that when W is N, L 2 and L 3 are not (L2FF), (L2MM), (L2NN), (L2OO), or (L2PP).
  • L2FF L2FF
  • L2MM L2MM
  • L2NN L2NN
  • L2OO L2PP
  • the present disclosure provides an antibody-drug conjugate of the fifth through the twentieth embodiments, wherein:
  • L 2 and L 3 independently, are a connecting spacer selected from a group consisting of
  • # of L 2 or L 3 indicates the point of direct attachment to E 1 or E 2 , respectively, the ## of L 2 or L 3 indicates the point of direct attachment to W;
  • R L is hydrogen or -C(O)-R H ; and
  • d is an integer from 20 to 30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27,
  • the present disclosure provides an antibody-drug conjugate of the twenty-first embodiment, wherein d is 25.
  • the definitions of the remaining variables are provided in the twenty-first embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the twenty-second embodiments, wherein the peptide group comprises 1 to 4, 1 to 3, or 1 to 2 amino acid residues.
  • the definitions of the remaining variables are provided in the seventh through the twenty-second embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the twenty-third embodiment, wherein the amino acid residues are selected from glycine (Gly), L- valine (Vai), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (He), L- phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L- leucine (Leu), L-tryptophan (Trp), L-tyrosine (Tyr) and (3-alanine (
  • the definitions of the remaining variables are provided in the twenty-third embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through the twenty-third embodiments, wherein the peptide group comprises Val- Cit, Phe-Lys, Vai-Ala, Val-Lys, Leu-Cit, Cit-((3-Ala), Gly-Gly-Gly, Gly-Gly-Phe-Gly (SEQ ID NO: 37), and/or sulfo-Ala-Val-Ala.
  • the definitions of the remaining variables are provided in the first through the twenty-third embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the twenty-third through the twenty-fifth embodiments, wherein the peptide group represented by E 1 or E 2 is an enzyme cleavage element.
  • E 1 or E 2 is an enzyme cleavage element.
  • the definitions of the remaining variables are provided in the twenty-third through the twenty-fifth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the twenty-third through the twenty-fifth embodiments, or pharmaceutically acceptable salt thereof, wherein the peptide group represented by E 1 or E 2 is a hydrophilic moiety.
  • the definitions of the remaining variables are provided in the twenty-third through the twenty-fifth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the twenty-sixth embodiment, or pharmaceutically acceptable salt thereof, wherein E 1 or E 2 , independently, is an enzyme cleavage element selected from a group consisting of wherein A of E 1 or E 2 indicates the point of direct attachment to V 1 or V 2 in Formula (B) or direct attachment to the -NH- group in Formula (C) and (D); and AA of E 1 or E 2 indicates the point of direct attachment to L 2 or L 3 , respectively.
  • E 1 or E 2 independently, is an enzyme cleavage element selected from a group consisting of wherein A of E 1 or E 2 indicates the point of direct attachment to V 1 or V 2 in Formula (B) or direct attachment to the -NH- group in Formula (C) and (D); and AA of E 1 or E 2 indicates the point of direct attachment to L 2 or L 3 , respectively.
  • the definitions of the remaining variables are provided in the twenty-sixth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the twenty-seventh embodiment, or pharmaceutically acceptable salt thereof, wherein E 1 or E 2 , independently, is a hydrophilic moiety represented by wherein R E is a hydrophilic group R H .
  • E 1 or E 2 independently, is a hydrophilic moiety represented by wherein R E is a hydrophilic group R H .
  • R E is a hydrophilic group R H .
  • the present disclosure provides an antibody-drug conjugate of the twenty-ninth embodiment, or pharmaceutically acceptable salt thereof, wherein each hydrophilic group R H in E 1 or E 2 is independently ; wherein e is an integer between 20 and 30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30).
  • e is an integer between 20 and 30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30).
  • the definitions of the remaining variables are provided in the twenty-ninth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the thirtieth embodiment, wherein e is 24.
  • e is 24.
  • the definitions of the remaining variables are provided in the thirtieth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-first embodiments, or pharmaceutically acceptable salt
  • a 1 and A 2 independently are a bond or OH , wherein * indicates the point of attachment to D 1 or D 2 .
  • a 1 and A 2 are as
  • a 1 and A 2 are OH ;
  • a 2 is a bond; bond and A 2 is
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-second embodiments, wherein A 1 and A 2 are a bond.
  • a 1 and A 2 are a bond.
  • the definitions of the remaining variables are provided in the seventh through the thirty-second embodiments or any embodiments described therein.
  • X is a bond, triazolyl, or -CH 2 -triazolyl-, wherein X is connected to R 2 or R 3 ; or
  • X is -CH 2 -triazolyl-Ci-4 alkylene-OC(O)NHS(O) 2 NH-,
  • X is a bond, triazolyl, or -CH 2 -triazolyl-
  • R M5 in each occurrence, is independently H or Cmalkyl.
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-fifth embodiments, or pharmaceutically acceptable salt thereof, wherein L 4 and L 5 are each independently a spacer moiety selected from a group consisting of
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-sixth embodiments, wherein the hydrophilic groups represented by R 2 and R 3 each independently comprises polyethylene glycol, polyalkylene glycol, a polyol, a polysarcosine, a sugar, an oligosaccharide, a polypeptide, C2-C6 alkyl substituted with 1 to 3
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-seventh embodiments, wherein R 2 or R 3 independently is
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-eighth embodiments, wherein the hydrophilic group represented by R 2 or R 3 each independently comprises:
  • R 23 is H, -CH 3 or -C 11 2 C 11 2 C( O )011 ; or
  • the hydrophilic group represented by R 2 or R 3 each independently comprises: a polysarcosine with the following moiety: wherein f is an integer between 3 and 25; and
  • R 23 is H, -CH 3 or -C 11 2 C 11 2 C( O )011.
  • the definitions of the remaining variables are provided in the seventh through the thirty-eighth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-sixth embodiments, wherein the enzyme cleavage element represented by R 2 or R 3 each independently comprises:
  • the present disclosure provides an antibody-drug conjugate of any one of the seventh through the thirty-sixth embodiments, wherein R 2 or R 3 , independently, is selected from a group consisting of wherein g and h are independently an integer between 20 and 30.
  • the present disclosure provides an antibody-drug conjugate of the thirty-ninth through the forty-first embodiments, wherein g is 23, 24, or 25; and h is 23, 24, or 25.
  • the present disclosure provides an antibody-drug conjugate of the seventh embodiment, wherein the dual linker is represented by the following formula:
  • a 2 i g for each occurrence is independently an integer between 20 and 30 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30); o for each occurrence is independently an integer between 1 and 9 (e.g., between 2 and 5); n is an integer between 1 and 12 (e.g., between 2 and 5); indicates the point of attachment to the Ab; and indicates the point of direct attachment to D 1 or D 2 .
  • a 1 and A 2 are both bond.
  • the present disclosure provides an antibody-drug conjugate of the seventh embodiment, wherein the dual linker is represented by Formula (D5):
  • the present disclosure provides an antibody-drug conjugate of the first embodiment, or pharmaceutically acceptable salt thereof, wherein the dual linker is represented by the following formula:
  • D 1 and D 2 are each independently a BH3 mimetic.
  • one of DI and D2 is a BH3 mimetic selected from a Mcl-1 inhibitor, a Bcl-2 inhibitor, and a Bcl-xL inhibitor, and the other is an antineoplastic non-BH3 mimetic selected from topoisomerase 1 inhibitor or an anti-mitotic drug.
  • the definitions of the remaining variables are provided in the second through forty-fifth embodiments or any embodiments described therein.
  • D 1 is a BH3 mimetic and D 2 is an antineoplastic non-BH3 mimetic; and the definitions of the remaining variables are provided in the second through forty- fifth embodiments or any embodiments described therein.
  • D 1 is selected from a Mcl-1 inhibitor, a Bcl-2 inhibitor, and a Bcl-xL inhibitor, and D 2 is a topoisomerase 1 inhibitor or an anti-mitotic drug.
  • D 1 is a Bcl-xL inhibitor and D 2 is a topoisomerase 1 inhibitor.
  • D 1 is a Bcl-xL inhibitor and D 2 is an anti-mitotic drug.
  • D 1 and/or D 2 are each independently selected from a Mcl-1 inhibitor, a Bcl-2 inhibitor and a Bcl-xL inhibitor. .
  • the present disclosure provides an antibody-drug conjugate of any one of the second through forty-sixth embodiments, wherein D 1 and D 2 are both (i) a Mcl-1 inhibitor; (ii) a Bcl-2 inhibitor; or (iii) Bcl-xL inhibitor.
  • D 1 and D 2 are both (i) a Mcl-1 inhibitor; (ii) a Bcl-2 inhibitor; or (iii) Bcl-xL inhibitor.
  • the definitions of the remaining variables are provided in the second through forty-sixth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through forty-seventh embodiments, wherein D 1 and D 2 are the same.
  • the definitions of the remaining variables are provided in the second through forty-sixth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through forty-seventh embodiments, wherein D 1 and D 2 are different.
  • the definitions of the remaining variables are provided in the second through forty-seventh embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through forty-seventh embodiments, or pharmaceutically acceptable salt thereof, wherein (i) one of D 1 and D 2 is a Mcl-1 inhibitor and the other is a Bcl-2 inhibitor; (ii) one of D 1 and D 2 is a Mcl-1 inhibitor and the other is a Bcl-xL inhibitor; or (iii) one of D 1 and D 2 is a Bcl-2 inhibitor and the other is a Bcl-xL inhibitor.
  • the definitions of the remaining variables are provided in the second through forty-seventh embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the second through fortyseventh embodiments, or pharmaceutically acceptable salt thereof, wherein (i) D 1 is a Mcl-1 inhibitor and D 2 is a Mcl-1 inhibitor; (ii) D 1 is a Mcl-1 inhibitor and D 2 is a Bcl-2 inhibitor; (iii) D 1 is a Bcl-xL inhibitor and D 2 is a Bcl-xL inhibitor: (iv) D 1 is a Bcl-xL inhibitor and D 2 is a Bcl-2 inhibitor; or (v) D 1 is a Bcl-2 inhibitor and D 2 is a Mcl-1 inhibitor; or (vi) D 1 is a Mcl-1 inhibitor and D 2 is a Bcl-xL inhibitor.
  • the definitions of the remaining variables are provided in the second through forty-seventh embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the forty-sixth through fiftieth embodiments, or pharmaceutically acceptable salt thereof, the Mcl-1 inhibitor is represented by Formula (I):
  • Ring D 0 is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group
  • Ring E 0 is a furyl, thienyl or pyrrolyl ring
  • X 01 , X 03 , X 04 and X 05 independently of one another are a carbon atom or a nitrogen atom
  • X 02 is a C-R 026 group or a nitrogen atom
  • ⁇ means that the ring is aromatic
  • Y 0 is a nitrogen atom or a C-R 03 group
  • Z0 is a nitrogen atom or a C-R04 group
  • R 01 is a halogen atom, a linear or branched (C 1 -C 6 )alkyl group, a linear or branched (C2-C6)alkenyl group, a linear or branched (C2-C6)alkynyl group, a linear or branched (C1- C6)haloal
  • the present disclosure provides an antibody-drug conjugate of the fifty-first embodiment, wherein Cy 01 , Cy 02 , Cy 03 , Cy 04 , Cy 05 , Cy 06 , Cy 07 , Cy 08 and Cy 010 , independently of one another, is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted by one or more groups selected from halo; - (C 1 -C 6 )alkoxy; -(C 1 -C 6 )haloalkyl; -(C 1 -C 6 )haloalkoxy; -(CH 2 ) p0 -O-SO 2 -OR 030 ; -(CH 2 ) p0 -SO 2 -OR 030 ;
  • the present disclosure provides an antibody-drug conjugate of the fifty-first embodiment, wherein the Mcl-1 inhibitor is presented by Formula (IA): wherein: Z0 is a nitrogen atom or a C-R04 group, R01 is a halogen atom, a linear or branched (C1-C6)alkyl group, a linear or branched (C2-C6)alkenyl group, a linear or branched (C2-C6)alkynyl group, a linear or branched (C1- C6)haloalkyl group, a hydroxy group, a linear or branched (C1-C6)alkoxy group, a -S-(C1- C6)alkyl group, a cyano group, -Cy08, -NR011R011’, R 02 , R 03 and R 04 independently of one another are a hydrogen
  • the present disclosure provides an antibody-drug conjugate of the fifty-first embodiment, wherein the Mcl-1 inhibitor is represented by Formula (IB):
  • R01 is a linear or branched (C1-C6)alkyl group
  • R03 is -O-(C1-C6)alkyl-NR011R011’, or , wherein R011 and R011’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (C1-C6)alkyl group, or -(C0-C6)alkyl-Cy01; or the pair (R011, R011’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (C 1 -C 6 )alkyl group, and wherein R 027 is a hydrogen atom and R 028 is a -(CH2)p0-O-SO2-O
  • the definitions of the remaining variables are provided in the fifty-first embodiment.
  • the present disclosure provides an antibody-drug conjugate of the fifty-fourth embodiment, wherein R 01 is methyl or ethyl.
  • the definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the fifty-fourth embodiment, wherein R03 is -O-CH2-CH2-NR011R011’ in which R011 and R011’ form, together with the nitrogen atom carrying them, a piperazinyl group which may be substituted by a group being a hydrogen atom or a linear or branched (C1-C6)alkyl group).
  • R03 is -O-CH2-CH2-NR011R011’ in which R011 and R011’ form, together with the nitrogen atom carrying them, a piperazinyl group which may be substituted by a group being a hydrogen atom or a linear or branched (C1-C6)alkyl group).
  • the present disclosure provides an antibody-drug conjugate of the fifty-fourth embodiment, wherein R 03 comprises the formula: , wherein R027 is a hydrogen atom and R028 is a - (CH2)p0-SO2-OR030 group. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • R03 comprises the formula: , wherein is a bond to the linker. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the fifty-fourth embodiment, wherein R09 is Cy02. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the fifty-ninth embodiment, wherein Cy 02 is an optionally substituted aryl group. The definitions of the remaining variables are provided in the fifty-ninth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the fifty-fourth embodiment, wherein Cy 05 comprises a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • Cy 05 comprises a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • Cy 05 is a pyrimidinyl group. The definitions of the remaining variables are provided in the fifty-fourth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the fifty-fourth through sixty-second embodiments, wherein the Mcl-1 inhibitor is attached by a covalent bond to R 03 of formula (I), (IA), or (IB); or is attached by a covalent bond to R 09 of formula (I), (IA), or (IB).
  • the definitions of the remaining variables are provided in the fifty-fourth through sixty-second embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the fifty-fourth through sixty-third embodiments, wherein the Mcl-1 inhibitor is represented by any one of the following formulas: Table A1
  • the present disclosure provides an antibody-drug conjugate of any one of the forty-sixth through fiftieth embodiments, wherein the Bcl-xL inhibitor is represented by Formula (II) or Formula (III): , or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing, wherein: R1 and R2 independently of one another represent a group selected from the group consisting of: hydrogen; a linear or branched C1-C6alkyl optionally substituted by a hydroxyl or a C1- C6alkoxy group; a C3-C6cycloalkyl; a trifluoromethyl; and a linear or branched C1- C6alkylene-heterocycloalkyl wherein the heterocycloalkyl group is optionally substituted by a linear or branched C1-C6alkyl group; or R1 and R2
  • A1 is –NH-, -N(C1-C3alkyl), O, S or Se
  • A2 is N, CH or C(R5)
  • RG4 is selected from the group consisting of hydrogen, a C1-C6alkyl optionally substituted by 1 to 3 halogen atoms, a C1-C6alkyl substituted by a hydroxyl, a C1-C6alkyl substituted by a C1- C6alkoxy group, a C2-C6alkenyl, a C2-C6alkynyl and a C3-C6cycloalkyl, and RG5 represents a hydrogen atom or a C1-C6alkyl group optionally substituted by 1 to 3 halogen atoms, R 4 represents a hydrogen, fluorine, chlorine or bromine atom, a methyl, a hydroxyl or a methoxy group, R5 represents a group selected from the group consisting of: a C1-C6alkyl optionally substituted by 1 to 3 halogen atoms; a C 2 -C 6 alkenyl; a C 2 -C 6
  • the present disclosure provides an antibody-drug conjugate of the sixty-fifth embodiment, wherein the Bcl-xL inhibitor is represented by formula (IIA) or (IIIA):
  • R3 represents a group selected from the group consisting of: hydrogen; a C3-C6cycloalkyl; a linear or branched C1-C6alkyl; -X1-NRaRb; -X1-N + RaRbRc; -X1-O-Rc; -X1-N3 and , Ra and Rb independently of one another represent a group selected from the group consisting of: hydrogen; a linear or branched C1-C6alkyl optionally substituted by one or two hydroxyl groups; and a C 1 -C 6 alkylene-SO 2 O-, R c represents a hydrogen or a linear or branched C 1 -C 6 alkyl group, Het 2 represents a group selected from the group consisting of: , A
  • Cy represents a C3-C8cycloalkyl
  • R8 represents a group selected from the group consisting of: hydrogen; a linear or branched C1-C6alkyl, -NR’aR’b; -NR’a-CO-OR’c; -NR’a-CO-R’c; -N + R’aR’bR’c; -O-R’c; -NH-X’2- N +R’aR’bR’c; -O-X’2-NR’aR’b; -X’2-NR’aR’b; -NR’c-X’2-N3 and:
  • R10 represents a group selected from the group consisting of hydrogen, fluorine, chlorine, bromine, -CF 3 and methyl
  • R 11 represents a group selected from the group consisting of hydrogen, a C 1 -C 3 alkylene-R 8 , - O-C 1 -C 3 alkylene-R 8 , -CO-NR h R
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through sixty-seventh embodiments, wherein R 7 represents a group selected from the group consisting of: a linear or branched C 1 -C 6 alkyl group; a (C 3 -C 6 )cycloalkylene-R 8 ; wherein Cy represents a C3-C8cycloalkyl.
  • R 7 represents a group selected from the group consisting of: a linear or branched C 1 -C 6 alkyl group; a (C 3 -C 6 )cycloalkylene-R 8 ; wherein Cy represents a C3-C8cycloalkyl.
  • the definitions of the remaining variables are provided in the sixty-fifth through sixty-seventh embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through sixty-seventh embodiments, wherein R7 represents a group selected from the group consisting of: .
  • R7 represents a group selected from the group consisting of: .
  • the definitions of the remaining variables are provided in the sixty-fifth through sixty-seventh embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the sixty-fifth embodiment, wherein the Bcl-xL inhibitor is represented by formula (IIB), (IIC), (IIIB) or (IIIC):
  • R3 represents a group selected from: hydrogen; linear or branched C1-C6alkyl; -X1-NRaRb; -X1-N + RaRbRc; and -X1-O-Rc;
  • Z1 represents a bond
  • R3 represents hydrogen; or Z1 represents –O-
  • R3 represents –X1-NRaRb, Ra and Rb independently of one another represent a group selected from: hydrogen; linear or branched C 1 -C 6 alkyl optionally substituted by one or two hydroxyl groups; and C 1 - C 6 alkylene-SO 2 O-
  • R c represents a hydrogen or a linear or branched C 1 -C 6 alkyl group
  • R 6 represents –X 2 -O-R 7
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through seventieth embodiments, wherein R 7 represents the following group: .
  • the definitions of the remaining variables are provided in the sixty-fifth through seventieth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through seventieth embodiments, wherein R7 represents a group selected .
  • the definitions of the remaining variables are provided in the sixty-fifth through seventieth embodiments or any embodiments described therein .
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through seventy-second embodiments, wherein R 8 represents a group selected from: wherein represents a bond to the linker
  • R 8 represents a group selected from: wherein represents a bond to the linker
  • the definitions of the remaining variables are provided in the sixty-fifth through seventy-second embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the sixty-fifth through seventy-third embodiments, wherein B3 represents a C3- C8heterocycloalkyl group selected from a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a morpholinyl group, an azepanyl group, and a 4,4-difluoropiperidin-1-yl group.
  • B3 represents a C3- C8heterocycloalkyl group selected from a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a morpholinyl group, an azepanyl group, and a 4,4-difluoropiperidin-1-yl group.
  • the definitions of the remaining variables are provided in the sixty-fifth through seventy-third embodiments or any embodiments described therein .
  • the present disclosure provides an antibody-drug
  • the present disclosure provides an antibody-drug conjugate of any one of the forty-sixth through fiftieth embodiments, or pharmaceutically acceptable salt thereof, wherein the Bcl-2 inhibitor is represented by Formula (IV) or Formula (V): (IV), or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing, wherein: A1 represents a hydrogen or halogen atom, a linear or branched (C1-C6)polyhaloalkyl group, a linear or branched (C1-C6)alkyl group or a cycloalkyl group, A2 represents a linear or branched (C1-C
  • aryl means a phenyl, naphthyl, biphenyl or indenyl group
  • heteroaryl means any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary nitrogens)
  • cycloalkyl means any mono- or bi-cyclic, non-aromatic, carbocyclic group containing from 3 to 10 ring members
  • heterocycloalkyl means any mono- or bi-cyclic, non-aromatic, condensed or spiro group composed of from 3 to 10 ring members and containing from 1 to 3 hetero atoms selected from oxygen, sulphur, SO, SO 2 and nitrogen, and it is possible for the aryl, hetero
  • the present disclosure provides an antibody-drug conjugate of the seventy-sixth or seventy-seventh embodiment, wherein, in Formula (IV), (i) A1 represents a hydrogen atom or a methyl group; or (ii) A1 and A2 both represent a methyl group.
  • the definitions of the remaining variables are provided in the seventy-sixth or seventy-seventh embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the seventy-sixth through seventy-eighth embodiments, wherein, in Formula (IV), T represents a methyl, aminomethyl, (morpholin-4-yl)methyl, (4-methylpiperazin-1-yl)methyl, 2-(morpholin-4- yl)ethyl [2 (morpholin 4 yl)ethoxy]methyl hydroxymethyl [2 (dimethylamino)ethoxy]methyl, hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-ylmethyl, 1-oxa-6-azaspiro[3.3]hept-6-ylmethyl, 3- (morpholin-4-yl)propyl or trifluoromethyl group.
  • T represents a methyl, aminomethyl, (morpholin-4-yl)methyl, (4-methylpiperazin-1-yl)methyl, 2-(morpholin-4- yl)ethyl [2 (morpholin 4 yl)
  • R 3 represents a group selected from phenyl, 1H-pyrazole, 1H-indole, 1H-indazole, pyridine, pyrimidine, 1H- pyrrolo[2,3-b]pyridine, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridine, 1H-benzimidazole, 1H-pyrrole, 1H- pyrrolo[2,3-c]pyridine, 1H-pyrrolo[3,2-b]pyridine, 5H-pyrrolo[3,2-d]pyrimidine, thiophene, pyrazine, 1H-pyrazolo[3,
  • the present disclosure provides an antibody-drug conjugate of the seventy-sixth embodiment, wherein the Bcl-2 inhibitor is represented by Formula (V) or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • the definitions of the remaining variables are provided in the seventy-sixth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the seventy-sixth embodiment, wherein the Bcl-2 inhibitor is represented by Formula (Va): or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • the definitions of the remaining variables are provided in the seventy-sixth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the eighty-first or eighty-second embodiment, wherein R3 in Formula (V) or (Va) represents the following group: and R c represents a group selected from: hydrogen, linear or branched (C 1 -C 6 )alkyl group optionally substituted by 1 to 3 halogen atoms, (C 1 -C 6 )alkylene-NR d R e , (C 1 -C 6 )alkylene-OR j , cycloalkyl, heterocycloalkyl, and (C 1 -C 6 )alkylene-heterocycloalkyl group.
  • R 3 in Formula (V) or (Va) represents the following group: and R c represents a group selected from: hydrogen, linear or branched (C 1 -C 6 )alkyl group optionally substituted by 1 to 3 halogen atoms, (C 1 -C 6 )alkylene-NR d R e , (C
  • the present disclosure provides an antibody-drug conjugate of the eighty-third embodiment, wherein Rc represents a methyl group.
  • the definitions of the remaining variables are provided in the eighty-third embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of any one of the eighty-first through eighty-third embodiments, wherein R 4 in Formula (V) or (Va) represents the following group: .
  • the definitions of the remaining variables are provided in the eighty-first through eighty-third embodiments or any embodiments described therein.
  • the present disclosure provides a process antibody-drug conjugate of the eighty-first embodiment, wherein the Bcl-2 inhibitor is represented by Formula (Vb):
  • the present disclosure provides an antibody-drug conjugate of the eighty-sixth embodiment, wherein R c in Formula (Vb) represents a methyl group.
  • R c in Formula (Vb) represents a methyl group.
  • the definitions of the remaining variables are provided in the eighty-sixth embodiment or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate of the eighty-first embodiment, wherein the Bcl-2 inhibitor is represented by Formula (Vc), (Vd),
  • the present disclosure provides an antibody-drug conjugate of any one of the eighty-first through eighty-eighth embodiments, wherein in Formula (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi) or (Vj): (i) X represents a bond; (ii) A1 represents C-Y4; (iii) R a and R b both represent a hydrogen atom; (iv) R 5 represents a hydrogen atom, a hydroxy group or a fluorine atom, preferably a hydroxy group; (v) R 6 represents a hydrogen atom, or a fluorine atom, preferably a hydrogen
  • the Bcl-2 inhibitor is represented by Formula (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi) or (Vj), wherein: (i) X represents a bond; (ii) A 1 represents C-Y 4 ; (iii) R a and R b both represent a hydrogen atom; (iv) R 5 represents a hydrogen atom, a hydroxy group or a fluorine atom, preferably a hydroxy group; (v) R 6 represents a hydrogen atom, a fluorine atom, preferably a hydrogen atom; (vi) A1 represents C-H and Y2 represents a hydrogen atom; (vii) Y1 and Y5 represent both a hydrogen atom, or: Y1 and
  • R5 represents a hydroxy group and R6 represents a hydrogen atom.
  • R6 represents a hydrogen atom.
  • Y3 represents a -O-(C1-C4)alkylene- Cy3 group.
  • the present disclosure provides an antibody-drug conjugate described in any one of the first to forty-sixth, forty-ninth, fifty-first to ninieth embodiments, wherein the topoisomerase 1 inhibitor is represented by any one of the following or a pharmaceutically acceptable salt thereof: Table A4
  • the definitions of the remaining variables are provided in any one of the first to forty-sixth, forty- ninth, fifty-first to ninieth embodiments or any embodiments described therein.
  • the present disclosure provides an antibody-drug conjugate described in any one of the first to forty-sixth, forty-ninth, fifty-first to ninieth embodiments, wherein anti- mitotic drug is monomethyl auristatin E (MMAE) or a taxane.
  • MMAE monomethyl auristatin E
  • the definitions of the remaining variables are provided in any one of the first to forty-sixth, forty-ninth, fifty-first to ninieth embodiments or any embodiments described therein.
  • the taxane is selected from docetaxel, paclitaxel, or cabazitaxel.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody or antigen-binding fragment thereof is an anti-CD7 antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) and three light chain CDRs selected from the group consisting of a) heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3, light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6; b) heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:28, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:29, heavy chain CDR3 (HCDR3) consist
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody or antigen-binding fragment thereof is an anti-CD7 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-CD7 antibody or antigen- binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; and a light chain amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:17 or a sequence that is at least 95% identical to SEQ ID NO:17, and a light chain amino acid sequence of SEQ ID NO:14 or a sequence that is at least 95% identical to SEQ ID NO:14.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibodycomprising a heavy chain amino acid sequence of SEQ ID NO:18 or a sequence that is at least 95% identical to SEQ ID NO:18, and a light chain amino acid sequence of SEQ ID NO:14 or a sequence that is at least 95% identical to SEQ ID NO:14.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:19 or a sequence that is at least 95% identical to SEQ ID NO:19, and a light chain amino acid sequence of SEQ ID NO:14 or a sequence that is at least 95% identical to SEQ ID NO:14.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20, and a light chain amino acid sequence of SEQ ID NO:15 or a sequence that is at least 95% identical to SEQ ID NO:15.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:21 or a sequence that is at least 95% identical to SEQ ID NO:21, and a light chain amino acid sequence of SEQ ID NO:15 or a sequence that is at least 95% identical to SEQ ID NO:15.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:22 or a sequence that is at least 95% identical to SEQ ID NO:22, and a light chain amino acid sequence of SEQ ID NO:15 or a sequence that is at least 95% identical to SEQ ID NO:15.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:23 or a sequence that is at least 95% identical to SEQ ID NO:23, and a light chain amino acid sequence of SEQ ID NO:15 or a sequence that is at least 95% identical to SEQ ID NO:15.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20, and a light chain amino acid sequence of SEQ ID NO:16 or a sequence that is at least 95% identical to SEQ ID NO:16.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24, and a light chain amino acid sequence of SEQ ID NO:16 or a sequence that is at least 95% identical to SEQ ID NO:16.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody is an anti-CD7 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24, and a light chain amino acid sequence of SEQ ID NO:15 or a sequence that is at least 95% identical to SEQ ID NO:15.
  • the present disclosure provides an antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody or antigen binding fragment thereof is an -anti-CD7 antibody or antigen-binding fragment thereof comprising 1) one or more cysteine substitutions selected from E152C, S375C, or both E152C and S375C of the heavy chain of the antibody or antigen binding fragment thereof; or 2) one or more cysteine substitutions selected from V205C, S400C, or both V205C and S400C of the heavy chain of the antibody or antigen binding fragment thereof; wherein the position is numbered according to the EU system.
  • the present disclosure provides antibody-drug conjugate of any one of the first through ninetieth embodiments, wherein the antibody or antigen binding fragment thereof is an anti-CD7 antibody or antigen-binding fragment thereof comprising one or more Fc silencing mutations.
  • the present disclosure provides, in part, novel antibody-drug conjugate (ADC) compounds with biological activity against cancer cells.
  • ADC antibody-drug conjugate
  • the compounds may slow, inhibit, and/or reverse tumor growth in mammals, and/or may be useful for treating human cancer patients.
  • the present disclosure more specifically relates, in some embodiments, to ADC compounds that are capable of binding and killing cancer cells.
  • the ADC compounds disclosed herein comprise a dual linker that attaches two BH3 mimetics to a full-length antibody or an antigen- binding fragment.
  • the ADC compounds are also capable of internalizing into a target cell after binding [124]
  • D1 and/or D2 in the ADC compounds disclosed herein e.g., ADCs of Formula (A), (B), (C), (D1), (D2), or (D3) in the present disclosure
  • Table A1a
  • D1 and/or D2 in the ADC compounds disclosed herein independently comprises a formula selected from any one of the formulae in Table A2a, or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • Table A2a
  • D1 and/or D2 in the ADC compounds disclosed herein independently comprises a formula selected from any one of the formulae in Table A3a, or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • Table A3a
  • D1 and/or D2 in the ADC compounds disclosed herein independently comprises a formula selected from any one of the formulae in TableA4a, or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • Table A4a wherein represents a bond to the linker.
  • the maleimide group the compound of Table B form a covalent bond with the antibody or antigen-binding fragment thereof (Ab) to form the ADC compound of formula (A) comprising moiety, wherein * indicates the connection point to Ab.
  • these compounds can contain one pharmaceutically acceptable monovalent anionic counterion M1-.
  • the monovalent anionic counterion M1- can be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like.
  • the monovalent anionic counterion M 1 - is trifluoroacetate or formate.
  • the ADCs depicted above can also be represented by the following formula: wherein represents an anti-CD7 antibody or an antigen fragment thereof covalently linked to the linker-payload (L/P) depicted above; a is an integer from 1 to 16. In some embodiments, a is an integer from 1 to 8. In some embodiments, a is an integer from 1 to 5. In some embodiments, a is an integer from 2 to 4. In some embodiments, a is 2. In some embodiments, a is 4. In some embodiments, a is determined by liquid chromatography-mass spectrometry (LC-MS).
  • LC-MS liquid chromatography-mass spectrometry
  • the antibody is an anti-CD7 antibody or antigen fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to a target antigen CD7 on a cancer cell.
  • P-L-P refers to the linker-payloads, linker-drugs, or linker-compounds disclosed herein and the terms “P#-L#-P#” refers to a specific dual linker-drug disclosed herein, wherein each of the codes “P#” refers to a specific antineoplastic compound (e.g. BH3 mimetics) unless otherwise specified and L# refers to a specific dual linker unless otherwise specified.
  • the two “P#” codes can be the same or different, i.e. refers to the same or different antineoplastic compounds (e.g. BH3 mimetics).
  • P1-L1-P1 refers to the linker-payload compound with dual linker L1 attaches to two P1 payloads
  • P1-L1-P2 refers to the linker-payload compound with dual linker L1 attaches to a P1 and a P2 payload, including an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or pharmaceutically acceptable salt of any of the foregoing.
  • the terms "P1-L1-P2" and “P2-L1-P1” refer to two different linker-drugs.
  • “L#-P#” refer to a specific mono linker-drug disclosed herein.
  • “L1- P1” refers to the linker-payload compound with mono linker L1 attaches to one P1 payload.
  • the antibody or antigen-binding fragment binds to a target antigen on a cancer cell.
  • the target antigen is CD7.
  • the antibody or antigen-binding fragment are antibodies or antigen- binding fragments disclosed in international application publication WO2018/098306, which is incorporated by reference in it entirety.
  • compositions comprising multiple copies of an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein).
  • the average p of the antibody-drug conjugates in the composition is from about 2 to about 4.
  • compositions comprising an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein) or a composition (e.g., any of the exemplary compositions described herein), and a pharmaceutically acceptable carrier.
  • an antibody-drug conjugate e.g., any of the exemplary antibody-drug conjugates described herein
  • a composition e.g., any of the exemplary compositions described herein
  • a pharmaceutically acceptable carrier e.g., any of the exemplary compositions described herein
  • therapeutic uses for the described ADC compounds and compositions e.g., in treating a cancer.
  • the present disclosure provides methods of treating a cancer (e.g., a cancer that expresses an antigen targeted by the antibody or antigen-binding fragment of the ADC, such as CD7 ).
  • the present disclosure provides methods of reducing or slowing the expansion of a cancer cell population in a subject. In some embodiments, the present disclosure provides methods of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an ADC compound or composition disclosed herein.
  • An exemplary embodiment is a method of treating a subject having or suspected of having a cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein).
  • the cancer expresses a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein).
  • the tumor expresses a target antigen CD7.
  • the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, or spleen cancer.
  • the tumor is a gastric cancer.
  • administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.
  • Another exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein).
  • the cancer cell population expresses a target antigen CD7.
  • the cancer cell population is from a tumor or a hematological cancer.
  • the cancer cell population is from a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or
  • the cancer cell population is from a lymphoma or gastric cancer.
  • administration of the antibody- drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.
  • administration of the antibody-drug conjugate, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.
  • Another exemplary embodiment is an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) for use in treating a subject having or suspected of having a cancer.
  • the cancer expresses a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • Another exemplary embodiment is a use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) in treating a subject having or suspected of having a cancer.
  • the cancer expresses a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • Another exemplary embodiment is a use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) in a method of manufacturing a medicament for treating a subject having or suspected of having a cancer.
  • the cancer expresses a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • Another exemplary embodiment is a method of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) by providing a biological sample from the subject; contacting the sample with the antibody-drug conjugate; and detecting binding of the antibody-drug conjugate to cancer cells in the sample.
  • the cancer cells in the sample express a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.
  • An exemplary embodiment is a method of producing an antibody-drug conjugate byconjuating an anti-CD7 antibody or antigen-binding fragment to a dual linker joined or covalently attached to two antineoplastic compounds, wherein at least one antineoplastic compound is a BH3 mimetic (e.g., two BH3 mimetics or a BH3 mimetic and a non-BH3 mimetic (e.g., topoisomerase I inhibitor)) under conditions that allow conjugation.
  • a BH3 mimetic e.g., two BH3 mimetics or a BH3 mimetic and a non-BH3 mimetic (e.g., topoisomerase I inhibitor)
  • FIG. 1 shows the in vitro activity of anti-CD7 BclxL ADC high DAR and anti-CD7 dual Bcl2/BclxL ADC in J45.01 (TALL) cell line as single agent or in combination with ABT199 (CTG 72h).
  • FIG. 2 shows the in vitro activity of anti-CD7 BclxL ADC high DAR and anti-CD7 dual Bcl2/BclxL in BE.13 (TALL) cell line as single agent or in combination with ABT199 (CTG 72h).
  • FIG. 3 shows the in vitro activity of anti-CD7 BclxL ADC high DAR and anti-CD7 dual Bcl2/BclxL ADC in HNT-34 (AML) cell line as single agent or in combination with ABT199 (CTG 72h).
  • FIG. 4 is a graph showing tumor volume (mm3) over time (day) of BE13-grafted female NOD- SCID mice upon treatment with Ab N - P5-L12-P5 (CD7-targeting ADC Fc-silent) and Ab G1_P5-L12- P5 (
  • compositions and methods are described more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. [151] Throughout this text, the descriptions refer to compositions and methods of using the compositions.
  • an antibody drug conjugate is referred to as “Target X-P1-L1-P2”, such a conjugate would comprise an antibody that binds Target X, a dual linker designated as L1, and two payloads designated as P1 and P2, respectively.
  • an antibody drug conjugate is referred to as “anti-Target X-P1- L1-P2”, such a conjugate would comprise an antibody that binds Target X, a dual linker designated as L1, and two payloads designated as P1 and P2, respectively.
  • an antibody drug conjugate is referred to as “AbX-P1-L1-P2”
  • such a conjugate would comprise the antibody designated as AbX, a dual linker designated as L1, and two payloads designated as P1 and P2, respectively.
  • a control antibody drug conjugate comprising a non-specific, isotype control antibody may be referenced as “isotype control IgG1-P1-L1-P2” or “IgG1-P1-L1-P2”.
  • Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.
  • Isotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, and 36 Cl. Accordingly, it should be understood that the present disclosure includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as 3 H and 14 C, or those into which non-radioactive isotopes, such as 2 H and 13 C are present.
  • Such isotopically labelled compounds are useful in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F or labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, e.g., using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
  • the term “about” refers to a range of values which are 10% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 1% more or less than the specified value.
  • antibody-drug conjugate refers to one or more therapeutic compounds (e.g., an antineoplastic payload, such as a BH3 mimetic moiety, a topoisomerase 1 inhibitor, or an anti-mitotic drug) that is linked to one or more antibodies or antigen-binding fragments.
  • antineoplastic payload such as a BH3 mimetic moiety, a topoisomerase 1 inhibitor, or an anti-mitotic drug
  • antineoplastic payloads e.g. BH3 mimetic moieties, topoisomerase 1 inhibitors, or anti-mitotic drugs
  • “2a” refers to the number of antineoplastic payloads (e.g.
  • antibody is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • An antibody can be polyclonal or monoclonal, multiple or single chain, or an intact immunoglobulin, and may be derived from natural sources or from recombinant sources.
  • An “intact” antibody is a glycoprotein that typically comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • An antibody can be a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody.
  • the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass.
  • An antibody can be an intact antibody or an antigen-binding fragment thereof.
  • the antibody or antibody fragment disclosed herein include modified or engineered amino acid residues, e.g., one or more cysteine residues, as sites for conjugation to a drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932).
  • the disclosure provides a modified antibody or antibody fragment comprising a substitution of one or more amino acids with cysteine at the positions described herein.
  • Sites for cysteine substitution are in the constant regions of the antibody or antibody fragment and are thus applicable to a variety of antibody or antibody fragment, and the sites are selected to provide stable and homogeneous conjugates.
  • a modified antibody or fragment can have one, two or more cysteine substitutions, and these substitutions can be used in combination with other modification and conjugation methods as described herein.
  • a modified antibody comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 191, 195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 and 422 of a heavy chain of the antibody, and wherein the positions are numbered according to the EU system.
  • a modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system, and wherein the light chain is a human kappa light chain.
  • a modified antibody or antibody fragment thereof comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions at positions 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, or position 107 of an antibody light chain and wherein the positions are numbered according to the EU system.
  • a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine on its constant regions wherein the substitution is position 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, position 107 of an antibody light chain, position 165 of an antibody light chain or position 159 of an antibody light chain and wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain.
  • a modified antibody or antibody fragment thereof comprises a combination of substitution of two amino acids with cysteine on its constant regions wherein the combinations comprise substitutions at positions 375 of an antibody heavy chain and position 152 of an antibody heavy chain, wherein the positions are numbered according to the EU system.
  • a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 360 of an antibody heavy chain, wherein the positions are numbered according to the EU system.
  • a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 107 of an antibody light chain and wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain.
  • antibody fragment or “antigen-binding fragment” or “functional antibody fragment,” as used herein, refers to at least one portion of an antibody that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen (e.g., CD7).
  • Antigen-binding fragments may also retain the ability to internalize into an antigen- expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity.
  • the terms antibody, antibody fragment, antigen-binding fragment, and the like, are intended to embrace the use of binding domains from antibodies in the context of larger macromolecules such as ADCs.
  • fragments of a full-length antibody can perform the antigen binding function of a full- length antibody.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen-binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, bispecific or multi-specific antibody constructs, ADCs, v-NAR and bis-scFv (see, e.g., Holliger and Hudson (2005) Nat Biotechnol.23(9):1126-36).
  • Antigen-binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see US Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • scFv refers to a fusion protein comprising at least one antigen-binding fragment comprising a variable region of a light chain and at least one antigen-binding fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • Antigen- binding fragments are obtained using conventional techniques known to those of skill in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as are intact antibodies.
  • Antigen-binding fragments for example, may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage.
  • CDR complementarity determining region
  • HCDR1, HCDR2, and HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, and LCDR3 three CDRs in each light chain variable region
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed.
  • the CDRs correspond to the amino acid residues that are defined as part of the Kabat CDR, together with the amino acid residues that are defined as part of the Chothia CDR.
  • the CDRs defined according to the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) (e.g., insertion(s) after position 35), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1) (e.g., insertion(s) after position 27), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1) (e.g., insertion(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1) (e.g., insertion(s) after position 30), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs comprise or consist of, e.g., amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.
  • the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).
  • the CDR regions of an antibody may be determined using the program IMGT/DomainGap Align.
  • the term "monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., US Patent No.4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J Mol Biol.222:581-97, for example.
  • the term also includes preparations of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the monoclonal antibodies described herein can be non-human, human, or humanized.
  • the term specifically includes "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.
  • the term “human antibody,” as used herein, refers an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.
  • the term includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al. ((2000) J Mol Biol.296(1):57- 86).
  • immunoglobulin variable domains e.g., CDRs
  • CDRs may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia, and/or ImMunoGenTics (IMGT) numbering.
  • the human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).
  • human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • recombinant human antibody refers to a human antibody that is prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • chimeric antibody refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable regions of both heavy and light chains correspond to the variable regions of antibodies derived from one species with the desired specificity, affinity, and activity while the constant regions are homologous to antibodies derived from another species (e.g., human) to minimize an immune response in the latter species.
  • humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are a type of chimeric antibody which contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (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), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody can be further modified by the substitution of residues, either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or activity.
  • an Fc region refers to a polypeptide comprising the CH3, CH2 and at least a portion of the hinge region of a constant domain of an antibody.
  • an Fc region may include a CH4 domain, present in some antibody classes.
  • An Fc region may comprise the entire hinge region of a constant domain of an antibody.
  • an antibody or antigen-binding fragment comprises an Fc region and a CH1 region of an antibody.
  • an antibody or antigen-binding fragment comprises an Fc region CH3 region of an antibody.
  • an antibody or antigen-binding fragment comprises an Fc region, a CH1 region, and a kappa/lambda region from the constant domain of an antibody.
  • an antibody or antigen-binding fragment comprises a constant region, e.g., a heavy chain constant region and/or a light chain constant region.
  • a constant region is modified compared to a wild-type constant region. That is, the polypeptide may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or to the light chain constant region domain (CL).
  • Example modifications include additions, deletions, or substitutions of one or more amino acids in one or more domains. Such changes may be included to optimize effector function, half-life, etc.
  • Internalizing refers to an antibody or antigen-binding fragment that is capable of being taken through the cell’s lipid bilayer membrane to an internal compartment (i.e., “internalized”) upon binding to the cell, preferably into a degradative compartment in the cell.
  • an internalizing anti-CD7 antibody is one that is capable of being taken into the cell after binding to CD7 on the cell membrane.
  • the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., CD7) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC transfers through the cellular membrane after antigen binding).
  • the internalizing antibody or antigen-binding fragment binds a receptor on the cell surface.
  • An internalizing antibody or internalizing antigen-binding fragment that targets a receptor on the cell membrane may induce receptor- mediated endocytosis.
  • the internalizing antibody or internalizing antigen-binding fragment is taken into the cell via receptor-mediated endocytosis.
  • Non-internalizing as used herein in reference to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that remains at the cell surface upon binding to the cell.
  • the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen-binding fragment (i.e., the ADC remains at the cell surface and does not transfer through the cellular membrane after antigen binding).
  • cluster of differentiation 7 or “CD7,” as used herein, refers to any native form of human CD7.
  • the term encompasses full-length human CD7 (e.g., NCBI Reference Sequence: NP_006128.1; SEQ ID NO:25), as well as any form of human CD7 that may result from cellular processing.
  • the term also encompasses functional variants or fragments of human CD7, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CD7 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only).
  • CD7 can be isolated from human, or may be produced recombinantly or by synthetic methods.
  • anti-CD7 antibody or “antibody that binds to CD7,” as used herein, refers to any form of antibody or antigen-binding fragment thereof that binds, e.g., specifically binds, to CD7.
  • the term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments so long as they bind, e.g., specifically bind, to CD7.
  • WO2018/098306 provides and is incorporated herein by reference for exemplary CD7-binding sequences, including exemplary anti-CD7 antibody sequences.
  • the anti-CD7 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antigen-binding fragment.
  • binding specificity refers to the ability of an individual antibody or antigen binding fragment to preferentially react with one antigenic determinant over a different antigenic determinant. The degree of specificity indicates the extent to which an antibody or fragment preferentially binds to one antigenic determinant over a different antigenic determinant.
  • the term “specific,” “specifically binds,” and “binds specifically” refers to a binding reaction between an antibody or antigen-binding fragment (e.g., an anti-CD7 antibody) and a target antigen (e.g., CD7) in a heterogeneous population of proteins and other biologics.
  • Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 2, 5, 7, 10 or more times more affinity than to the irrelevant antigen or antigen mixture, then it is considered to be specific.
  • a “specific antibody” or a “target-specific antibody” is one that only binds the target antigen (e.g., CD7), but does not bind (or exhibits minimal binding) to other antigens.
  • an antibody or antigen-binding fragment that specifically binds a target antigen (e.g., CD7) has a K D of less than 1x10 -6 M, less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M, less than 1x10- 10 M, less than 1x10 -11 M, less than 1x10 -12 M, or less than 1x10 -13 M.
  • the K D is 1 pM to 500 pM.
  • the K D is between 500 pM to 1 ⁇ M, 1 ⁇ M to 100 nM, or 100 mM to 10 nM.
  • affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Without being bound by theory, within each antigen binding site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, typically the stronger the affinity.
  • the binding affinity of an antibody is the sum of the attractive and repulsive forces operating between the antigenic determinant and the binding site of the antibody.
  • the term "kon” or “ka” refers to the on-rate constant for association of an antibody to the antigen to form the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.
  • the term “koff” or “kd” refers to the off-rate constant for dissociation of an antibody from the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.
  • KD refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. KD is calculated by ka/kd.
  • epitopes refers to the portion of an antigen capable of being recognized and specifically bound by an antibody (or antigen-binding fragment). Epitope determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide.
  • An epitope may be “linear” or “conformational.” Conformational and linear epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the epitope bound by an antibody may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen- antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen.
  • Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligo- peptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) BioDrugs 21:145- 56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56). [183] Competitive binding and epitope binning can also be used to determine antibodies sharing identical or overlapping epitopes.
  • competitive binding can be evaluated using a cross-blocking assay, such as the assay described in “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory, Harlow and Lane (1 st edition 1988, 2 nd edition 2014).
  • a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as CD7 (e.g., a binding protein comprising CDRs and/or variable domains selected from those identified in Tables 3-5), by at least about 50% in the cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between), and/or vice versa.
  • competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance where antibodies or binding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol.66, pp.55-66)).
  • competitive binding can be used to sort groups of binding proteins that share similar epitopes. For example, binding proteins that compete for binding can be “binned” as a group of binding proteins that have overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or nearby epitopes.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the terms encompass amino acid polymers comprising two or more amino acids joined to each other by peptide bonds, amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally-occurring amino acid, as well as naturally-occurring amino acid polymers and non-naturally-occurring amino acid polymers.
  • the terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a "recombinant” protein refers to a protein (e.g., an antibody) made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid.
  • An "isolated” protein refers to a protein unaccompanied by at least some of the material with which it is normally associated in its natural state.
  • an "isolated antibody,” as used herein, is an antibody that has been identified and separated from one or more (e.g., the majority) of the components (by weight) of its source environment, e.g., from the components of a hybridoma cell culture or a different cell culture that was used for its production.
  • the separation is performed such that it sufficiently removes components that may otherwise interfere with the suitability of the antibody for the desired applications (e.g., for therapeutic use).
  • Methods for preparing isolated antibodies include, without limitation, protein A chromatography, anion exchange chromatography, cation exchange chromatography, virus retentive filtration, and ultrafiltration.
  • variant refers to a nucleic acid sequence or an amino acid sequence that differs from a reference nucleic acid sequence or amino acid sequence respectively, but retains one or more biological properties of the reference sequence.
  • a variant may contain one or more amino acid substitutions, deletions, and/or insertions (or corresponding substitution, deletion, and/or insertion of codons) with respect to a reference sequence. Changes in a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid sequence, or may result in amino acid substitutions, additions, deletions, fusions, and/or truncations.
  • a nucleic acid variant disclosed herein encodes an identical amino acid sequence to that encoded by the unmodified nucleic acid or encodes a modified amino acid sequence that retains one or more functional properties of the unmodified amino acid sequence.
  • a variant of a nucleic acid or peptide can be a naturally-occurring variant or a variant that is not known to occur naturally. Variants of nucleic acids and peptides may be made by mutagenesis techniques, by direct synthesis, or by other techniques known in the art. A variant does not necessarily require physical manipulation of the reference sequence.
  • a variant has high sequence identity (i.e., 60% nucleic acid or amino acid sequence identity or higher) as compared to a reference sequence.
  • a peptide variant encompasses polypeptides having amino acid substitutions, deletions, and/or insertions as long as the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity with a reference sequence, or with a corresponding segment (e.g., a functional fragment) of a reference sequence, e.g., those variants that also retain one or more functions of the reference sequence.
  • a corresponding segment e.g., a functional fragment
  • a nucleic acid variant encompasses polynucleotides having amino acid substitutions, deletions, and/or insertions as long as the polynucleotide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity with a reference sequence, or with a corresponding segment (e.g., a functional fragment) of a reference sequence.
  • the term “conservatively modified variant” applies to both amino acid and nucleic acid sequences.
  • nucleic acid sequences conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • conservatively modified variants include individual substitutions, deletions, or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar amino acids are well known in the art.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of, e.g., an antibody or antigen-binding fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antigen-binding fragment by standard techniques known in the art, such as, e.g., site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested using the functional assays described herein.
  • the term “homologous” or “identity,” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are matched or homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the output is the percent identity of the subject sequence with respect to the query sequence.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • amino acid identity or homology between proteins disclosed herein and variants thereof, including variants of target antigen CD7 and variants of antibody variable domains (including individual variant CDRs) is at least 80% to the sequences depicted herein, e.g., identities or homologies of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100%, or 100%.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J Mol Biol. 48:444- 53) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • An exemplary set of parameters is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • agent is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, an extract made from biological materials, or a combination of two or more thereof.
  • therapeutic agent or “drug” refers to an agent that is capable of modulating a biological process and/or has biological activity.
  • the BH3 mimetics and the ADCs comprising them, as described herein, are exemplary therapeutic agents.
  • chemotherapeutic agent or “anti-cancer agent” is used herein to refer to all agents that are effective in treating cancer (regardless of mechanism of action). Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent.
  • Chemotherapeutic agents include antibodies, biological molecules, and small molecules, and encompass the BH3 mimeticand ADCs comprising them, as described herein.
  • a chemotherapeutic agent may be a cytotoxic or cytostatic agent.
  • cytostatic agent refers to an agent that inhibits or suppresses cell growth and/or multiplication of cells.
  • cytotoxic agent refers to a substance that causes cell death primarily by interfering with a cell’s expression activity and/or functioning.
  • antineoplastic payload refers to a compound or compounds that slow or inhibit the division of cancerous cells or that kill the cancerous cells.
  • antineoplastic payloads include BH3 mimetic compounds (e.g., MC1-1 inhibitors, Bcl-xL inhibitors, or Bcl-2 inhibitors), topoisomerase 1 inhibitors (e.g., topotecan, exatecan, deruxtecan or SN-38) or anti-mitotic drugs (e.g., monomethyl auristatin E (MMAE) or a taxane).
  • the antineoplastic payload is a BH3 mimetic compound.
  • the antineoplastic payload is a topoisomerase 1 inhibitor.
  • the antineoplastic payload is an anti-mitotic drug.
  • antineoplastic non-BH3 mimetic refers to a compound or compounds that are not BH3 mimetics and slow or inhibit the division of cancerous cells or that kill the cancerous cells.
  • antineoplastic non-BH3 mimetic include topoisomerase 1 inhibitors (e.g., topotecan, exatecan, deruxtecan or SN-38) or anti-mitotic drugs (e.g., monomethyl auristatin E (MMAE) or a taxane).
  • MMAE monomethyl auristatin E
  • the antineoplastic non-BH3 mimetic is a topoisomerase 1 inhibitor.
  • the antineoplastic non-BH3 mimetic is an anti-mitotic drug.
  • BH3 mimetic refers to an agent capable of disrupting the interaction between the proapoptotic and antiapoptotic members of the Bcl-2 family and are potent inducers of apoptosis.
  • exemplary BH3 mimetics include inhibitors of Bcl-2, Bcl-xL, Bcl-w and Mcl-1.
  • Mcl-1 myeloid cell leukemia 1
  • Mcl-1 myeloid cell leukemia 1
  • the term encompasses full-length human Mcl-1 (e.g., UniProt Reference Sequence: Q07820), as well as any form of human Mcl-1 that may result from cellular processing.
  • Mcl-1 can be isolated from human, or may be produced recombinantly or by synthetic methods.
  • inhibitor means to reduce a biological activity or process by a measurable amount, and can include but does not require complete prevention or inhibition.
  • “inhibition” means to reduce the expression and/or activity of BH3 mimetic and/or one or more upstream modulators or downstream targets thereof.
  • Mcl-1 inhibitor refers to an agent capable of reducing the expression and/or activity of Mcl-1 and/or one or more upstream modulators or downstream targets thereof.
  • Exemplary Mcl-1 modulators (including exemplary inhibitors of Mcl-1) are described in WO 2015/097123; WO 2016/207216; WO 2016/207217; WO 2016/207225; WO 2016/207226; WO 2017/125224; WO 2019/035899, WO 2019/035911, WO 2019/035914, WO 2019/035927, US 2019/0055264, WO 2016/033486, WO 2017/147410, WO 2018/183418, and WO 2017/182625, each of which are incorporated herein by reference as exemplary Mcl- 1 modulators, including exemplary Mcl- 1 inhibitors, that can be included as drug moieties in the disclosed ADCs.
  • Mcl-1 inhibitors that can be included as drug moieties in the disclosed ADCs are those of formula: wherein each variable is defined as in W02019/035911; WO 2019/035899; WO 2019/035914; or WO
  • Mcl-1 inhibitor drug moiety refers to the component of an ADC or composition that provides the structure of an Mcl- 1 inhibitor compound or a compound modified for attachment to an ADC that retains essentially the same, similar, or enhanced biological function or activity as compared to the original compound.
  • Mcl-1 inhibitor drug moiety is component (D 1 and/or D 2 ) in an ADC of Formula (A).
  • the Mcl-1 inhibitor is represented by Formula (I) described herein
  • the Mcl-1 inhibitor is a compound described in any one of the fiftieth through sixty-third embodiments in the summary section of the present disclosure.
  • B-cell lymphoma-extra large refers to any native form of human Bcl-xL, an anti-apoptotic member of the Bcl-2 protein family.
  • the term encompasses full-length human Bcl-xL (e.g, UniProt Reference Sequence: Q07817-1), as well as any form of human Bcl-xL that may result from cellular processing.
  • Bcl-xL can be isolated from human, or may be produced recombinantly or by synthetic methods.
  • Bcl-xL inhibitor refers to an agent capable of reducing the expression and/or activity of Bcl-xL and/or one or more upstream modulators or downstream targets thereof.
  • Exemplary Bcl-xL modulators are described in
  • a “Bcl-xL inhibitor drug moiety”, “Bcl-xL inhibitor”, and the like refer to the component of an ADC or composition that provides the structure of a Bcl-xL inhibitor compound or a compound modified for attachment to an ADC that retains essentially the same, similar, or enhanced biological function or activity as compared to the original compound.
  • Bcl-xL inhibitor drug moiety is component (D 1 and/or D 2 ) in an ADC of Formula (A).
  • the Bcl-xL inhibitor is represented by Formula (II) or Formula (III) described herein: some embodiments, the Bcl-xL inhibitor is a compound described in any one of the sixty-fourth through seventy-fourth embodiments in the summary section of the present disclosure.
  • B-cell lymphoma 2 refers to any native form of human Bcl-2, an anti-apoptotic member of the Bcl-2 protein family.
  • the term encompasses full-length human Bcl-2 (e.g, UniProt Reference Sequence: Pl 0415), as well as any form of human Bcl-2 that may result from cellular processing.
  • Mcl-1 can be isolated from human, or may be produced recombinantly or by synthetic methods.
  • Bcl-2 inhibitor refers to an agent capable of reducing the expression and/or activity of Bcl-2 and/or one or more upstream modulators or downstream targets thereof.
  • Exemplary Bcl-2 modulators are described in WO 2013/110890, WO 2015/011400, WO 2015/011399, WO 2015/011397, WO 2015/011396, WO 2015/011164 and WO 2019081559, each of which are incorporated herein by reference as exemplary Bcl- 2 modulators, including exemplary Bcl-2 inhibitors, that can be included as drug moieties in the disclosed ADCs.
  • a “Bcl-2 inhibitor drug moiety”, “Bcl-2 inhibitor”, and the like refer to the component of an ADC or composition that provides the structure of a Bcl-2 inhibitor compound or a compound modified for attachment to an ADC that retains essentially the same, similar, or enhanced biological function or activity as compared to the original compound.
  • Bcl-2 inhibitor drug moiety is component (D 1 and/or D 2 ) in an ADC of Formula (A).
  • the Bcl-2 inhibitor is represented by Formula (IV) or Formula (V) described herein: some embodiments, the Bcl-2 inhibitor is a compound described in any one of the seventy-fifth through eighty-ninth embodiments in the summary section of the present disclosure.
  • topoisomerase 1 inhibitor refers to a compound or compounds which interferes with the action of topoisomerase 1 enzyme.
  • agents include, but are not limited to, topotecan, exatecan, deruxtecan or SN-38..
  • anti-mitotic drug refers to a compound or compounds which targets mitosis regulating enzymes, such as mircrotubule regulating enzymes, Polo-like Kinases (PLK), Kinesin- Spindle Protein (KSP), Aurora kinases, and the like.
  • an anti-mitotic drug is monomethyl auristatin E (MMAE) or a taxane.
  • MMAE monomethyl auristatin E
  • taxane is selected from docetaxel, paclitaxel, or cabazitaxel.
  • cancer refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain morphological features. Often, cancer cells can be in the form of a tumor or mass, but such cells may exist alone within a subject, or may circulate in the blood stream as independent cells, such as leukemic or lymphoma cells.
  • cancer includes all types of cancers and cancer metastases, including hematological cancers, solid tumors, sarcomas, carcinomas and other solid and non-solid tumor cancers.
  • Hematological cancers may include B-cell malignancies, cancers of the blood (leukemias), cancers of plasma cells (myelomas, e.g., multiple myeloma), or cancers of the lymph nodes (lymphomas).
  • B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma.
  • Leukemias may include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • CMML chronic myelomonocytic leukemia
  • AoL acute monocytic leukemia
  • Lymphomas may include Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.
  • Other hematologic cancers may include myelodysplasia syndrome (MDS).
  • Solid tumors may include carcinomas such as adenocarcinoma, e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, etc.
  • carcinomas such as adenocarcinoma, e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, etc.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • the term “tumor” refers to any mass of tissue that results from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.
  • the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non- small cell lung cancer, prostate cancer, small cell lung cancer, pancreatic cancer, stomach cancer, colon cancer, head and neck cancer, or spleen cancer.
  • the tumor is a gastric cancer.
  • tumor cell and “cancer cell” may be used interchangeably herein and refer to individual cells or the total population of cells derived from a tumor or cancer, including both non- tumorigenic cells and cancer stem cells.
  • tumor cell and “cancer cell” will be modified by the term “non-tumorigenic” when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those cells from cancer stem cells.
  • target-negative refers to the absence of target antigen expression by a cell or tissue.
  • target-positive refers to the presence of target antigen expression.
  • a cell or a cell line that does not express a target antigen may be described as target-negative, whereas a cell or cell line that expresses a target antigen may be described as target-positive.
  • Non-human animals include all vertebrates (e.g., mammals and non- mammals) such as any mammal.
  • mammals include humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, and guinea pigs.
  • non-mammals include birds and fish.
  • the subject is a human.
  • a subject in need of treatment refers to a subject that would benefit biologically, medically, or in quality of life from a treatment (e.g. , a treatment with any one or more of the exemplary ADC compounds described herein).
  • treatment refers to any improvement of any consequence of disease, disorder, or condition, such as prolonged survival, less morbidity, and/or a lessening of side effects which result from an alternative therapeutic modality.
  • treatment comprises delaying or ameliorating a disease, disorder, or condition (i.e., slowing or arresting or reducing the development of a disease or at least one of the clinical symptoms thereof).
  • treatment comprises delaying, alleviating, or ameliorating at least one physical parameter of a disease, disorder, or condition, including those which may not be discernible by the patient.
  • treatment comprises modulating a disease, disorder, or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.
  • treatment comprises administration of a described ADC compound or composition to a subject, e.g., a patient, to obtain a treatment benefit enumerated herein.
  • the treatment can be to cure, heal, alleviate, delay, prevent, relieve, alter, remedy, ameliorate, palliate, improve, or affect a disease, disorder, or condition (e.g., a cancer), the symptoms of a disease, disorder, or condition (e.g., a cancer), or a predisposition toward a disease, disorder, or condition (e.g., a cancer).
  • a composition disclosed herein in addition to treating a subject having a disease, disorder, or condition, can also be provided prophylactically to prevent or reduce the likelihood of developing that disease, disorder, or condition.
  • the term “prevent”, “preventing,” or “prevention” of a disease, disorder, or condition refers to the prophylactic treatment of the disease, disorder, or condition; or delaying the onset or progression of the disease, disorder, or condition.
  • a "pharmaceutical composition” refers to a preparation of a composition, e.g., an ADC compound or composition, in addition to at least one other (and optionally more than one other) component suitable for administration to a subject, such as a pharmaceutically acceptable carrier, stabilizer, diluent, dispersing agent, suspending agent, thickening agent, and/or excipient.
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier, stabilizer, diluent, dispersing agent, suspending agent, thickening agent, and/or excipient.
  • the pharmaceutical compositions provided herein are in such form as to permit administration and subsequently provide the intended biological activity of the active ingredient(s) and/or to achieve a therapeutic effect.
  • the pharmaceutical compositions provided herein preferably contain no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • Pharmaceutically acceptable carriers may enhance or stabilize the composition or can be used to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers can include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the carrier may be selected to minimize adverse side effects in the subject, and/or to minimize degradation of the active ingredient(s).
  • An adjuvant may also be included in any of these formulations.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • Formulations for parenteral administration can, for example, contain excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated napthalenes.
  • excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, ethylene-vinyl acetate co-polymer particles, and surfactants, including, for example, polysorbate 20.
  • salts refers to a salt which does not abrogate the biological activity and properties of the compounds of the invention, and does not cause significant irritation to a subject to which it is administered.
  • examples of such salts include, but are not limited to: (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, poly
  • the antibody-drug conjugates (ADCs), linkers, payloads and linker-payloads described herein can contain a monovalent anionic counterion Mf. Any suitable anionic counterion can be used.
  • the monovalent anionic counterion is a pharmaceutically acceptable monovalent anionic counterion.
  • the monovalent anionic counterion Mf can be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, the monovalent anionic counterion Mf is trifluoroacetate or formate.
  • the term “therapeutically effective amount” or “therapeutically effective dose,” refers to an amount of a compound described herein, e.g. , an ADC compound or composition described herein, to effect the desired therapeutic result (i.e., reduction or inhibition of an enzyme or a protein activity, amelioration of symptoms, alleviation of symptoms or conditions, delay of disease progression, a reduction in tumor size, inhibition of tumor growth, prevention of metastasis).
  • a therapeutically effective amount does not induce or cause undesirable side effects.
  • a therapeutically effective amount induces or causes side effects but only those that are acceptable by a treating clinician in view of a patient’s condition.
  • a therapeutically effective amount is effective for detectable killing, reduction, and/or inhibition of the growth or spread of cancer cells, the size or number of tumors, and/or other measure of the level, stage, progression and/or severity of a cancer.
  • the term also applies to a dose that will induce a particular response in target cells, e.g., a reduction, slowing, or inhibition of cell growth.
  • a therapeutically effective amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • a therapeutically effective amount can also vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific amount may vary depending on, for example, the particular pharmaceutical composition, the subject and their age and existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • a therapeutically effective amount of an ADC may reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieve one or more symptoms.
  • prophylactically effective amount refers to an amount of a compound disclosed herein, e.g. , an ADC compound or composition described herein, that is effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • a prophylactically effective amount can prevent the onset of disease symptoms, including symptoms associated with a cancer.
  • p or “drug loading” or “drug: antibody ratio” or “drug-to-antibody ratio” or “DAR” refers to the number of drug moieties per antibody or antigen-binding fragment, i.e., drug loading, or the number of BH3 mimetic moieties per antibody or antigen-binding fragment (Ab) in ADCs of Formula (1).
  • antineoplastic compound e.g. a BH3 mimetic drug moiety, a topoisomerase 1 inhibitor or an anti-mitotic drug
  • p refers to the number of antineoplastic compounds (e.g.
  • one dual linker attaches two antineoplastic compounds (e.g. two BH3 mimetic drug moieties, or a BH3 mimetic and a non-BH3 mimetic (e.g., a topoisomerase 1 inhibitor or an anti-mitotic drug) to an antibody or antigen-binding fragment, therefore,/? is 2 if the antibody or antigen-binding fragment only links with one dual linker having two antineoplastic compounds (e.g.
  • average p refers to the average number of antineoplastic compounds (e.g. two BH3 mimetic drug moieties, or a BH3 mimetic and a non-BH3 mimetic (e.g., a topoisomerase 1 inhibitor or an anti-mitotic drug) per antibody or antigen-binding fragment, also referred to as “average drug loading.”
  • the antibody-drug conjugate (ADC) compounds of the present disclosure include those with anticancer activity.
  • the ADC compounds include an antibody or antigen-binding fragment conjugated (i.e., covalently attached by a dual linker) to two antineoplastic compounds, such as a BH3 mimetic drug moiety (e.g., a Mcl-1 inhibitor, a Bcl-2 inhibitor, or a Bcl-xL inhibitor or a combination thereof), a topoisomerase 1 inhibitor (e.g., topotecan, exatecan, deruxtecan or SN-38), or an anti-mitotic drug (e.g., monomethyl auristatin E (MMAE) or a taxane), wherein at least one antineoplastic compound is a BH3 mimetic drug moiety, and wherein the antineoplastic compound when not conjugated to an antibody or antigen-binding fragment has a cytotoxic or cytostatic effect.
  • a BH3 mimetic drug moiety
  • the BH3 mimetic drug moiety when not conjugated to an antibody or antigen-binding fragment is capable of reducing the expression and/or activity of a Bcl-2 family protein (e.g., Mcl-1, Bcl-2 and/or Bcl-xL) and/or one or more upstream modulators or downstream targets thereof.
  • a Bcl-2 family protein e.g., Mcl-1, Bcl-2 and/or Bcl-xL
  • the ADCs disclosed herein may provide potent anti-cancer agents.
  • the ADC may provide improved activity, better cytotoxic specificity, and/or reduced off-target killing as compared to the antineoplastic compound when administered alone.
  • the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and antineoplastic compounds in isolation, (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize drug loading and drug-to-antibody ratios; (iv) allow delivery, e.g.. intracellular delivery, of the antineoplastic compound via stable attachment to the antibody or antigen-binding fragment; (v) retain ADC stability as an intact conjugate until transport or delivery to a target site; (vi) minimize aggregation of the ADC prior to or after administration; (vii) allow for the therapeutic effect, e.g.
  • cytotoxic effect of the antineoplastic compound after cleavage or other release mechanism in the cellular environment; (viii) exhibit in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and antineoplastic compounds in isolation; (ix) minimize off-target killing by the antineoplastic compound; and/or (x) exhibit desirable pharmacokinetic and pharmacodynamics properties, formulatability, and toxicologic/immunologic profiles. Each of these properties may provide for an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14: 1605-13).
  • the ADC compounds of the present disclosure may selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or to tumor tissue.
  • the cytotoxic and/or cytostatic activity of the ADC is dependent on target antigen expression in a cell.
  • the disclosed ADCs are particularly effective at killing cancer cells expressing a target antigen while minimizing off-target killing.
  • the disclosed ADCs do not exhibit a cytotoxic and/or cytostatic effect on cancer cells that do not express a target antigen.
  • Exemplary CD7-expressing cancers include but are not limited to hematological cancer, such as multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma or spleen cancer, acute T-Cell leukemia (T-ALL) and peripheral T-cell lymphomas (Gomes-Silva et al.
  • hematological cancer such as multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia
  • ADC compounds comprising an antibody or antigenbinding fragment thereof (Ab) covalently linked to two antineoplastic payloads, such as a BH3 mimetic, a topoisomerase 1 inhibitor, or an anti-mitotic drug (D 1 and D 2 ) through a dual linker (L), wherein at least one antineoplastic payload is a BH3 mimetic, and wherein the dual linker has one attachment point connected to the antibody and two attachment points to the two antineoplastic payloads, such as BH3 mimetics, and wherein the two antineoplastic payloads, such as BH3 mimetics, can be the same or different.
  • antineoplastic payloads such as a BH3 mimetic, a topoisomerase 1 inhibitor, or an anti-mitotic drug (D 1 and D 2 )
  • L dual linker
  • the dual linker has one attachment point connected to the antibody and two attachment points to the two antineoplastic payloads, such as BH3 mimetics,
  • the antibody or antigenbinding fragment thereof targets a cancer cell.
  • the antibody or antigenbinding fragment is able to bind to a tumor-associated antigen (e.g., CD7), e.g., with high specificity and high affinity.
  • the antibody or antigen-binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell.
  • the ADCs internalize upon binding to a target cell, undergo degradation, and release the Bcl-xL inhibitor drug moiety to kill cancer cells.
  • the antineoplastic payloads such as BH3 mimetics, topoisomerase 1 inhibitor, or anti-mitotic drug, may be released from the antibody and/or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.
  • the antibody or antigen-binding fragment (Ab) of Formula (1) includes within its scope an antiCD? antibody or antigen-binding fragment that specifically binds to the target antigen CD7 on a cell.
  • the antibody or antigen-binding fragment (Ab) of Formula (1) includes within its scope an anti-CD7 antibody or antigen-binding fragment that specifically binds to the target antigen on a cancer cell (e.g., CD7).
  • the antibody or antigen-binding fragment may bind to the target antigen CD7 with a dissociation constant (KD) of ⁇ 1 mM, ⁇ 100 nM or ⁇ 10 nM, or any amount in between, as measured by, e.g., BIAcore® analysis.
  • KD dissociation constant
  • the KD is 1 pM to 500 pM. In some embodiments, the KD is between 500 pM to 1 pM, 1 pM to 100 nM, or 100 mM to 10 nM. In some embodiments, the anti-CD7 antibody is not Ab D. [235] In some embodiments, the anti-CD7 antibody or antigen-binding fragment is a four-chain antiCD? antibody (also referred to as an immunoglobulin or a full-length or intact antibody), comprising two heavy chains and two light chains. In some embodiments, the anti-CD7 antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin. In some embodiments, the anti-CD7 antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind the target cancer antigen and/or provide at least one function of the immunoglobulin.
  • the anti-CD7 antibody or antigen-binding fragment is an internalizing anti-CD7 antibody or internalizing antigen-binding fragment thereof.
  • the internalizing anti-CD7 antibody or internalizing antigen-binding fragment thereof binds to the target cancer antigen expressed on the surface of a cell and enters the cell upon binding.
  • the anti-neoplastic payload of the ADC is released from the anti-CD7 antibody or antigen-binding fragment of the ADC after the ADC enters and is present in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized), e.g., by cleavage, by degradation of the antibody or antigenbinding fragment, or by any other suitable release mechanism.
  • the anti-CD7 antibodies or antigen-binding fragments comprise mutations that mediate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, these mutations are known as Fc Silencing, Fc Silent, or Fc Silenced mutations.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • amino acid residues L234 and L235 of the IgGl constant region are substituted to A234 and A235 (also known as “LALA”).
  • amino acid residue N297 of the IgGl constant region is substituted to A297 (also known as “N297A”).
  • amino acid residues D265 and P329 of the IgGl constant region are substituted to A265 and A329 (also known as “DAP A”).
  • Other antibody Fc silencing mutations may also be used.
  • the Fc silencing mutations are used in combination, for example D265A, N297A and P329A (also known as “D ANAPA”).
  • the Fc silencing mutations are used in combination, for example L234A, L235E, G237A.
  • E152C, S375C, V205C and S400C according to EU numbering of the antibody heavy chain to facilitate conjugation to linker-drug moieties
  • they are designated as “CysMab”; or if the anti-CD7 antibody or antigen-binding fragment has been modified with Fc silencing mutations D265A, N297A and P329A of the IgGl constant region according to EU numbering, “DANAPA” is added to the anti-CD7 antibody name, or if the anti-CD7 antibody or antigen-binding fragment has been modified with Fc silencing mutations D265A and P329A of the IgGl constant region according to EU numbering, “DAPA” is added to the anti-CD7 antibody name.
  • Amino acid sequences of exemplary antibodies of the present disclosure, in addition to exemplary antigen targets, are set forth in Tables D1-D5.
  • the antibody or antigen-binding fragment of an ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above or a set of six CDRs from any set of heavy and light chain variable domains listed in the tables above.
  • the antibody or antigen-binding fragment of an ADC disclosed herein may comprise amino acid sequences that are conservatively modified and/or homologous to the sequences listed in the tables above, so long as the ADC retains the ability to bind to its target cancer antigen (e.g. , with a KD of less than
  • ADC 1x1 O' 8 M retains one or more functional properties of the ADCs disclosed herein (e.g., ability to internalize, bind to an antigen target, e.g., an antigen expressed on a tumor or other cancer cell, etc.).
  • the antibody or antigen-binding fragment of an ADC disclosed herein further comprises human heavy and light chain constant domains or fragments thereof.
  • the antibody or antigen-binding fragment of the described ADCs may comprise a human IgG heavy chain constant domain (such as an IgGl) and a human kappa or lambda light chain constant domain.
  • the antibody or antigen-binding fragment of the described ADCs comprises a human immunoglobulin G subtype 1 (IgGl) heavy chain constant domain with a human Ig kappa light chain constant domain.
  • the target antigen for an ADC is CD7.
  • the anti-CD7 antibody comprises (i) a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; and (ii) a light chain amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 95% identical to SEQ ID NO: 13, and the light chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 12, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.
  • the anti-CD7 antibody is milatuzumab, or an antigen-binding fragment thereof.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 11 or a sequence that is at least 95% identical to SEQ ID NO: 11, and the light chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12.
  • the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 95% identical to SEQ ID NO: 17, and the light chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 14, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 95% identical to SEQ ID NO: 18, and the light chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence of SEQ ID NO: 14, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 95% identical to SEQ ID NO: 19, and the light chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 14, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20, and the light chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:20 and the light chain amino acid sequence of SEQ ID NO: 15, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 or a sequence that is at least 95% identical to SEQ ID NO:21, and the light chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO: 15, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:22 or a sequence that is at least 95% identical to SEQ ID NO:22, and the light chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:22 and the light chain amino acid sequence of SEQ ID NO: 15, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 or a sequence that is at least 95% identical to SEQ ID NO:23, and the light chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO: 15, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20, and the light chain amino acid sequence of SEQ ID NO: 16 or a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:20 and the light chain amino acid sequence of SEQ ID NO: 16, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24, and the light chain amino acid sequence of SEQ ID NO: 16 or a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:24 and the light chain amino acid sequence of SEQ ID NO: 16, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.
  • the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24, and the light chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the anti-CD7 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:24 and the light chain amino acid sequence of SEQ ID NO: 15, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27.
  • the anti- CD7 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:26 and the light chain variable region amino acid sequence of SEQ ID NO:27, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:26 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.
  • the anti- CD7 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 9, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti- CD7 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:7 and the light chain variable region amino acid sequence of SEQ ID NO: 10, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti- CD7 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 8 and the light chain variable region amino acid sequence of SEQ ID NO: 10, or sequences that are at least 95% identical to the disclosed sequences.
  • the anti-CD7 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.
  • the anti-CD7 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • heavy chain CDR1 consisting of SEQ ID NO: 1
  • heavy chain CDR2 HCDR2
  • HCDR3 heavy chain CDR3
  • LCDR1 light chain CDR1
  • LCDR2 light chain CDR2
  • LCDR3 light chain CDR3
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO:2), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5), and LCDR3 (SEQ ID NO:6).
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:28, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:29, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:30; light chain CDR1 (LCDR1) consisting of SEQ ID NO:31, light chain CDR2 (LCDR2) consisting of SEQ ID NO:32, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • heavy chain CDR1 HCDR1
  • HCDR2 heavy chain CDR2
  • HCDR3 heavy chain CDR3
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:28), HCDR2 (SEQ ID NO:29), HCDR3 (SEQ ID NO:30); LCDR1 (SEQ ID NO:31), LCDR2 (SEQ ID NO:32), and LCDR3 (SEQ ID NO:6).
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:33, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:34, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • heavy chain CDR1 consisting of SEQ ID NO:33
  • heavy chain CDR2 HCDR2
  • HCDR3 heavy chain CDR3
  • LCDR1 light chain CDR1
  • LCDR2 light chain CDR2
  • LCDR3 light chain CDR3
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:33), HCDR2 (SEQ ID NO:34), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5), and LCDR3 (SEQ ID NO:6).
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:36, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO:2), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:35), LCDR2 (SEQ ID NO:36), and LCDR3 (SEQ ID NO:6).
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:28, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:29, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:30; light chain CDR1 (LCDR1) consisting of SEQ ID NO:31, light chain CDR2 (LCDR2) consisting of SEQ ID NO:32, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • heavy chain CDR1 HCDR1
  • HCDR2 heavy chain CDR2
  • HCDR3 heavy chain CDR3
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:28), HCDR2 (SEQ ID NO:29), HCDR3 (SEQ ID NO:30); LCDR1 (SEQ ID NO:31), LCDR2 (SEQ ID NO:32), and LCDR3 (SEQ ID NO:6).
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:33, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:34, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:36, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.
  • heavy chain CDR1 HCDR1
  • HCDR2 heavy chain CDR2
  • HCDR3 heavy chain CDR3
  • the anti-CD7 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of the anti-CD7 antibody or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:33), HCDR2 (SEQ ID NO:34), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:35), LCDR2 (SEQ ID NO:36), and LCDR3 (SEQ ID NO:6).
  • Residues in two or more polypeptides are said to "correspond” if the residues occupy an analogous position in the polypeptide structures.
  • Analogous positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on amino acid sequence or structural similarities. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment.
  • amino acid substitutions are of single residues. Insertions usually will be on the order of from about 1 to about 20 amino acid residues, although considerably larger insertions may be tolerated as long as biological function is retained (e.g., binding to a target antigen). Deletions usually range from about 1 to about 20 amino acid residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at a final derivative or variant. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions can be made in accordance with the following chart depicted as Table E.
  • variant antibody sequences typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen binding proteins as needed.
  • variants may be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed.
  • CD7-targeting antibodies such as Ab N provided particularly improved drug: antibody ratio, aggregation level, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, potency), minimized off-target killing, and/or treatment efficacy.
  • Improved treatment efficacy can be measured in vitro or in vivo, and may include reduced tumor growth rate and/or reduced tumor volume.
  • alternate antibodies to the same targets are used and provide at least some of the favorable functional properties described above (e.g., improved stability, improved tumor targeting, improved treatment efficacy, etc.). In some embodiments, some or all of these favorable functional properties are observed when the disclosed linkers and antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs, are conjugated to an alternate CD7-targeting antibody or antigen-binding fragment.
  • linkers and antineoplastic payloads such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs, are conjugated to an alternate CD7-targeting antibody or antigen-binding fragment.
  • the linker in an ADC is stable extracellularly in a sufficient manner to be therapeutically effective.
  • the linker is stable outside a cell, such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell).
  • the term “intact,” used in the context of an ADC, means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., the antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs).
  • “stable,” in the context of a linker or ADC comprising a linker, means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage in between) in a sample of ADC are cleaved (or in the case of an overall ADC are otherwise not intact) when the ADC is present in extracellular conditions.
  • the linkers and/or ADCs disclosed herein are stable compared to alternate linkers and/or ADCs with alternate linkers and/or antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs.
  • the ADCs disclosed herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.
  • Whether a linker is stable extracellularly can be determined, for example, by including an ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability may allow the ADC time to localize to target cancer cells and prevent the premature release of the drug moiety, which could lower the therapeutic index of the ADC by indiscriminately damaging both normal and cancer tissues.
  • the linker is stable outside of a target cell and releases the drug moiety from the ADC once inside of the cell, such that the drug can bind to its target.
  • an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage or alternate release mechanism.
  • Linkers may impact the physico-chemical properties of an ADC.
  • a linker may be "cleavable” or “non-cleavable” (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13).
  • Cleavable linkers are designed to release the drug moiety (e.g., a antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs) when subjected to certain environment factors, e.g., when internalized into the target cell, whereas non-cleavable linkers generally rely on the degradation of the antibody or antigen-binding fragment itself.
  • a drug moiety e.g., a antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs
  • alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation.
  • C 1 -C 6 alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • C 1 -C 6 alkyl groups include methyl (a C 1 alkyl), ethyl (a C 2 alkyl), 1-methylethyl (a C 3 alkyl), n-propyl (a C 3 alkyl), isopropyl (a C 3 alkyl), n-butyl (a C 4 alkyl), isobutyl (a C 4 alkyl), sec-butyl (a C 4 alkyl), tert-butyl (a C 4 alkyl), n-pentyl (a C 5 alkyl), isopentyl (a C 5 alkyl), neopentyl (a C 5 alkyl) and hexyl (a C 6 alkyl).
  • alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond.
  • C 2 - C 6 alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, which is attached to the rest of the molecule by a single bond.
  • C 2 - C 6 alkenyl groups include ethenyl (a C 2 alkenyl), prop-1-enyl (a C 3 alkenyl), but-1-enyl (a C 4 alkenyl), pent-1-enyl (a C 5 alkenyl), pent-4-enyl (a C 5 alkenyl), penta-1,4-dienyl (a C 5 alkenyl), hexa-1-enyl (a C6alkenyl), hexa-2-enyl (a C6alkenyl), hexa-3-enyl (a C6alkenyl), hexa-1-,4-dienyl (a C6alkenyl), hexa-1- ,5-dienyl (a C6alkenyl) and hexa-2-,4-dienyl (a C6alkenyl).
  • C2-C3alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to three carbon atoms, which is attached to the rest of the molecule by a single bond.
  • Non-limiting examples of "C2-C3alkenyl” groups include ethenyl (a C2alkenyl) and prop-1-enyl (a C3alkenyl).
  • alkylene refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing no unsaturation.
  • C1- C6alkylene refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms.
  • Non-limiting examples of “C1-C6alkylene” groups include methylene (a C1alkylene), ethylene (a C2alkylene), 1-methylethylene (a C3alkylene), n-propylene (a C3alkylene), isopropylene (a C3alkylene), n- butylene (a C4alkylene), isobutylene (a C4alkylene), sec-butylene (a C4alkylene), tert-butylene (a C4alkylene), n-pentylene (a C5alkylene), isopentylene (a C5alkylene), neopentylene (a C5alkylene), and hexylene (a C6alkylene).
  • alkenylene refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing at least one double bond.
  • C 2 -C 6 alkenylene refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to six carbon atoms.
  • C 2 -C 6 alkenylene groups include ethenylene (a C 2 alkenylene), prop-1-enylene (a C 3 alkenylene), but-1-enylene (a C 4 alkenylene), pent-1-enylene (a C 5 alkenylene), pent-4-enylene (a C 5 alkenylene), penta-1,4-dienylene (a C 5 alkenylene), hexa-1-enylene (a C 6 alkenylene), hexa-2-enylene (a C 6 alkenylene), hexa-3-enylene (a C 6 alkenylene), hexa-1-,4-dienylene (a C 6 alkenylene), hexa-1-,5-dienylene (a C 6 alkenylene) and hexa-2-,4-dienylene (a C 6 alkenylene).
  • C 2 -C 6 alkenylene refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to three carbon atoms.
  • Non-limiting examples of "C 2 -C 3 alkenylene” groups include ethenylene (a C 2 alkenylene) and prop-1-enylene (a C 3 alkenylene).
  • cycloalkyl or “C3-C8cycloalkyl,” as used herein, refers to a saturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring system.
  • Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane and adamantanyl.
  • Non-limiting examples monocyclic C 3 -C 8 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.
  • aryl refers to a phenyl, naphthyl, biphenyl or indenyl group.
  • heteroaryl refers any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary nitrogens).
  • cycloalkyl refers to any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, which may include fused, bridged or spiro ring systems.
  • Non-limiting examples of fused bicyclic or bridged ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, and bicyclo[2.2.2]octane.
  • Non-limiting examples monocyclic C3-C8cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.
  • heterocycloalkyl means any mono- or bi-cyclic non-aromatic carbocyclic group, composed of from 3 to 10 ring members, and containing from one to 3 hetero atoms selected from oxygen, sulphur, SO, SO2 and nitrogen, it being understood that bicyclic group may be fused or spiro type.
  • C3-C8heterocycloalkyl refers to heterocycloalkyl having 3 to 8 ring carbon atoms.
  • the heterocycloalkyl can have 4 to 10 ring members.
  • heteroarylene, cycloalkylene, heterocycloalkylene mean a divalent heteroaryl, cycloalkyl and heterocycloalkyl.
  • haloalkyl refers to a linear or branched alkyl chain substituted with one or more halogen groups in place of hydrogens along the hydrocarbon chain.
  • halogen groups suitable for substitution in the haloalkyl group include Fluorine, Bromine, Chlorine, and Iodine.
  • Haloalkyl groups may include substitution with multiple halogen groups in place of hydrogens in an alkyl chain, wherein said halogen groups can be attached to the same carbon or to another carbon in the alkyl chain.
  • the alkyl, alkenyl, alkynyl, alkoxy, amino, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl groups may be optionally substituted by 1 to 4 groups selected from optionally substituted linear or branched (C 1 -C 6 )alkyl, optionally substituted linear or branched (C 2 -C 6 )alkenyl group, optionally substituted linear or branched (C 2 -C 6 )alkynyl group, optionally substituted linear or branched (C 1 -C 6 )alkoxy, optionally substituted (C 1 -C 6 )alkyl-S-, hydroxy, oxo (or N-oxide where appropriate), nitro, cyano, -C(O)-OR 0 ’, -O-C(O)-R 0 ’, -C(O)-NR 0 ’R 0 ’’, -NR 0
  • polyoxyethylene refers to a linear chain, a branched chain or a star shaped configuration comprised of (OCH2CH2) groups.
  • PEG12 as used herein means that t is 12.
  • polyalkylene glycol refers to a linear chain, a branched chain or a star shaped configuration comprised of (O(CH2)m)n groups.
  • attachment group refers to a bivalent moiety which links the bridging spacer to the antibody or fragment thereof.
  • the attachment or coupling group is a bivalent moiety formed by the reaction between a reaction group and a functional group on the antibody or fragment thereof.
  • Non limiting examples of such bivalent moieties include the bivalent chemical moieties given in Table F and Table G provided herein.
  • attachment point refers to a location on the linker that is connected to an antibody or an antineoplastic payload.
  • the location is an atom, such as carbon, nitrogen, sulfur, or oxygen, where the linker connects with an antibody or an antineoplastic payload through a covalent bond.
  • bridging spacer refers to one or more linker components which are covalently attached together to form a bivalent moiety which links the branching moiety W to the attachment group.
  • Non-limiting examples of the bridging spacer include groups L1-1, L1-2, L1-3, L1-4, L1-5, and L1-6 described herein.
  • the term “branching moiety”, as used herein, refers to a chemical moiety that connects with three or more groups in the dual linker of the present disclosure.
  • the branching moiety is N or CR w ; wherein R w is H or C1-6alkyl.
  • the cleavable group is selected so that activation occurs at the desired site of action, which can be a site in or near the target cells (e.g., carcinoma cells) or tissues such as at the site of therapeutic action or antineoplastic payload activity.
  • cleavage may be enzymatic and exemplary enzymatically cleavable groups include natural amino acids or peptide sequences that end with a natural amino acid, and are attached at their carboxyl terminus to the linker.
  • a cleavable group comprises a pyrophosphate group, a phosphate group, a glucuronide group, a peptide group, and/or a self-immolative group.
  • the term “enzyme cleavage element”, as used herein, comprises an element that is susceptible to enzymatic cleavage.
  • Nonlimiting examples of the enzymatic cleavage include peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, or lipase induced cleavage.
  • the enzyme cleavage element in the present disclosure refers to a dipeptide group that can be cleaved by a peptidase.
  • the dipeptide group is selected from a group consisting of E1-1 and E1-2 described herein.
  • the enzyme cleavage element in the present disclosure comprises a sugar moiety that can be cleaved by a glucosidase, such as a glucuronide group.
  • the enzyme cleavage element in the present disclosure comprises a phosphate or pyrophosphate moiety that can be cleaved by phosphatases.
  • the enzyme cleavage element is represented by or , wherein A 1 and A 2 are as defined herein, indicates the point of attachment to E 1 or E 2 ; and indicates the point of attachment to D 1 or D 2 .
  • connecting spacer refers to one or more linker components which are covalently attached together to form a bivalent moiety which links the branching moiety W to the function moiety E1 or E2 which comprises an enzyme cleavage element or a hydrophilic moiety.
  • Nonlimiting examples of the connecting spacer include groups L2-1 through L2-30 described herein.
  • hydrophilic group refers to the group that has hydrophilic properties which increases the aqueous solubility of the dual linker is attached to the linker group of the present disclosure.
  • hydrophilic moiety refers to the moiety that comprises a functional group having a hydrophilic group attached thereto.
  • the functional group mentioned here refers to the bivalent peptide spacer described in the present disclosure.
  • spacer moiety refers to one or more linker components which are covalently attached together to form a moiety which links the self-immolative group to the hydrophilic group or an enzyme cleavage element.
  • spacer moiety refers to L 4 or L 5 as defined herein.
  • bivalent peptide spacer refers to bivalent linker comprising one or more amino acid residues covalently attached together to form a moiety which links the bridging spacer to the self immolative spacer or an enzyme cleavage element.
  • the one or more amino acid residues can be an residue of amino acids selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucune (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocysteine, and desmethyl pyrrolysine.
  • amino acids selected from alanine (Ala), cyste
  • a “bivalent peptide spacer” is a combination of 2 to four amino acid residues where each residue is independently selected from a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu),methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucune (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocy
  • linker component refers to a chemical moiety that is a part of the linker.
  • a linker component can be a chemical moiety which is readily formed by reaction between two reactive groups.
  • Non-limiting examples of such chemical moieties are given in Table F.
  • a wavy line ( ) indicates the point of attachment of the partial structure to the rest of the molecule.
  • the term “self-immolative spacer”, as used herein, refers to a moiety comprising one or more triggering groups (TG) which are activated by acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage, and after activation the protecting group is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of a leaving group.
  • TG triggering groups
  • Non-limiting examples of self-immolative spacer include: TG-Xa-LG, TG-Ya-LG , , , , , , and , wherein such groups can be optionally substituted, and wherein: TG is a triggering group; Xa is O, NH or S; Xb is O, NH, NCH3 or S; X c is O or NH; Y a is CH 2 , CH 2 O or CH 2 NH; Y b is CH 2 , O or NH; Y c is a bond, CH 2 , O or NH, and LG is a leaving group such as a Drug moiety (D) of the Linker-Drug group of the invention.
  • D Drug moiety
  • the self-immolative spacer connected to a drug moiety is a moiety having the following structure: or , wherein E 1 and/or E 2 is an enzyme cleavage element, and A 1 , A 2 , D 1 , D 2 , R 2 , R 3 , L 3 , and L 4 are as defined herein.
  • the self-immolative spacer is moiety having the structure or , wherein E 1 and/or E 2 is a bivalent peptide spacer, R 2 and/or R 3 is an enzyme cleavage element, and A 1 , A 2 , D 1 , D 2 , L 3 , and L 4 are as defined herein.
  • the term “self-immolative group,” as used herein, refer to a group that can generate a cascade of disassembling reactions leading to the temporally sequential release of a leaving group when the TG is activated and removed.
  • the self-immolative group is a group having the structure or wherein A 1 , A 2 , R 2 , R 3 , L 3 , and L 4 are as defined herein, indicates the point of attachment to E 1 or E 2 ; and indicates the point of attachment to D 1 or D 2 . C.
  • an intermediate which is the precursor of the linker moiety
  • the drug moiety e.g., BH3 mimetics, such as a Mcl-1 inhibitor, a Bcl-2 inhibitor and/or a Bcl-xL inhibitor; topoisomerase 1 inhibitors, such as topotecan, exatecan, deruxtecan or SN-38; or anti-mitotic drugs, such as monomethyl auristatin E (MMAE) or a taxane
  • BH3 mimetics such as a Mcl-1 inhibitor, a Bcl-2 inhibitor and/or a Bcl-xL inhibitor
  • topoisomerase 1 inhibitors such as topotecan, exatecan, deruxtecan or SN-38
  • anti-mitotic drugs such as monomethyl auristatin E (MMAE) or a taxane
  • reactive groups are used on the drug and/or the intermediate or linker.
  • the product of the reaction between the drug and the intermediate, or the derivatized drug is subsequently reacted with the antibody or antigen-binding fragment under conditions that facilitate conjugation of the drug and intermediate or derivatized drug and antibody or antigen-binding fragment.
  • the intermediate or linker may first be reacted with the antibody or antigen-binding fragment, or a derivatized antibody or antigen-binding fragment, and then reacted with the drug or derivatized drug.
  • a number of different reactions are available for covalent attachment of the drug moiety and/or linker moiety to the antibody or antigen-binding fragment.
  • non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a drug moiety to an amino (or carboxy) group on an antibody or antigen-binding fragment.
  • bifunctional agents such as dialdehydes or imidoesters may also be used to link the amino group on a drug moiety to an amino group on an antibody or antigen-binding fragment.
  • a BH3 mimetic e.g., a BH3 mimetic, a topoisomerase 1 inhibitor, or an anti-mitotic drug
  • Schiff base reaction This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent.
  • Isothiocyanates may also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present disclosure.
  • Examples of drug moieties that can be generated and linked to an antibody or antigen-binding fragment using various chemistries known to in the art include Mcl-1 inhibitors, Bcl-2 inhibitors, and Bcl-xL inhibitors, e.g., the Mcl-1 inhibitors, Bcl-2 inhibitors, and Bcl-xL inhibitors described and exemplified herein. Additional examples of drug moieties that can be generated and linked to an antibody or antigen- binding fragment using various chemistries known to in the art include topoisomerase 1 inhibitors or anti- mitotic drugs described and exemplified herein. a.
  • Suitable BH3 mimetic D1 and/or D2 may comprise a Mcl-1 inhibitor compound of the formulas (I), (IA), (IB), or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or addition salt thereof with a pharmaceutically acceptable acid or base. Additionally, the drug moiety may comprise any compounds of the Mcl-1 inhibitor (D) described herein. [325] As used herein, “atropisomers,” are stereoisomers arising because of hindered rotation about a single bond, where energy differences due to steric strain or other contributors create a barrier to rotation that is high enough to allow for isolation of individual conformers (Bringmann et al.
  • Atropisomers may be as follows: .
  • a preferred atropisomer may be (5Sa), also named (5aS).
  • a drug moiety of the disclosure may be any one of the compounds disclosed in International Patent Application Publication Nos.
  • BH3 mimetics of the disclosure may comprise a compound of Formula (I), (IA) or (IB), wherein the definitions of the variables depicted therein are described above [329]
  • Cy01, Cy02, Cy03, Cy04, Cy05, Cy06, Cy07, Cy08 and Cy010 independently of one another are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, wherein the optional substituents are selected from optionally substituted linear or branched (C1-C6)alkyl, optionally substituted linear or branched (C 2 -C 6 )alkenyl group, optionally substituted linear or branched (C 2 -C 6 )alkynyl group, optionally substituted linear or branched (C 1 -C 6 )alkoxy, optionally substituted (C 1 -C 6 )alky
  • BH3 mimetics D1 and/or D2 of the disclosure may comprise any one of the following: , , , ,
  • the BH3 mimetics D1 and/or D2 comprise a formula selected from Table A1 or A1a or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or a pharmaceutically acceptable salt of any of the foregoing. b.
  • Suitable BH3 mimetics D1 and/or D2 may comprise a Bcl-xL inhibitor compound of the formulas (II), (IIA), (IIB), (IIC), (III), (IIIA), (IIIB) or (IIIC) or an enantiomer, diastereoisomer, and/or addition salt thereof with a pharmaceutically acceptable acid or base.
  • the BH3 mimetics D 1 and/or D 2 may comprise any compounds of the Bcl-xL inhibitors described herein.
  • the BH3 mimetics D1 and/or D2 comprise a formula selected from Table A2 or A2a.
  • the BH3 mimetics D1 and/or D2 comprise Bcl-xL inhibitor known in the art, for example, ABT-737 and ABT-263.
  • the BH3 mimetics D1 and/or D2 comprise a Bcl-xL inhibitor selected from:
  • Suitable BH3 mimetics D1 and/or D2 may comprise a Bcl-2 inhibitor compound of the formulas (IV) or (V) or an enantiomer, diastereoisomer, and/or addition salt thereof with a pharmaceutically acceptable acid or base. Additionally, the BH3 mimetics D 1 and/or D 2 may comprise any compounds of the Bcl-2 inhibitor described herein. [338] In some embodiments, the Bcl-2 inhibitor is represented by Formula (IV) or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • aryl means a phenyl, naphthyl, biphenyl or indenyl group
  • heteroaryl means any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary nitrogens)
  • cycloalkyl means any mono- or bi-cyclic, non-aromatic, carbocyclic group containing from 3 to 10 ring members
  • - heterocycloalkyl means any mono- or bi-cyclic, non-aromatic, condensed or spiro group composed of from 3 to 10 ring members and containing from 1 to 3 hetero atoms selected from oxygen, sulphur, SO, SO2 and nitrogen, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl
  • A1 represents a hydrogen atom or a methyl group.
  • A1 and A2 both represent a methyl group.
  • T represents a methyl, aminomethyl, (morpholin-4- yl)methyl, (4-methylpiperazin-1-yl)methyl, 2-(morpholin-4-yl)ethyl, [2-(morpholin-4-yl)ethoxy]methyl, hydroxymethyl, [2-(dimethylamino)ethoxy]methyl, hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)- ylmethyl, 1-oxa-6-azaspiro[3.3]hept-6-ylmethyl, 3-(morpholin-4-yl)propyl or trifluoromethyl group.
  • R3 represents a group selected from phenyl, 1H-pyrazole, 1H-indole, 1H-indazole, pyridine, pyrimidine, 1H-pyrrolo[2,3-b]pyridine, 2,3-dihydro-1H-pyrrolo[2,3- b]pyridine, 1H-benzimidazole, 1H-pyrrole, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[3,2-b]pyridine, 5H- pyrrolo[3,2-d]pyrimidine, thiophene, pyrazine, 1H-pyrazolo[3,4-b]pyridine, 1,2-oxazole, and pyrazolo[1,5-a]pyrimidine, those groups optionally having one or more substituents selected from halogen, linear or branched (C1-C6)alkyl, linear or branched (C1 C6)alkoxy,
  • R3 represents the following group: N R c N H 3 C and Rc represents a group selected from: hydrogen, linear or branched (C1-C6)alkyl group optionally substituted by 1 to 3 halogen atoms, (C1-C6)alkylene-NRdRe, (C1-C6)alkylene-ORj, cycloalkyl, heterocycloalkyl, and (C1-C6)alkylene-heterocycloalkyl group.
  • RC represents a methyl group.
  • R4 in Formula (V) or (Va) represents the following group: .
  • the Bcl-2 inhibitor is represented by Formula (Vb),. or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • RC represents a methyl group.
  • the Bcl-2 inhibitor is represented by Formula (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi) or (Vj) or an enantiomer, a diastereoisomer, and/or a pharmaceutically acceptable salt of any one of the foregoing.
  • the Bcl-2 inhibitor is represented by Formula (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi) or (Vj), wherein : (i) X represents a bond; (ii) A 1 represents C-Y 4 ; (iii) R a and R b represent both a hydrogen atom; (iv) R 5 represents a hydrogen atom, a hydroxy group or a fluorine atom, preferably a hydroxy group; (v) R 6 represents a hydrogen atom, a fluorine atom, preferably a hydrogen atom; (vi) A 1 represents C-H and Y 2 represents a hydrogen atom; (vii) Y 1 and Y 5 represent both a hydrogen atom, or: Y 1 and Y 5 represent a fluoro atom and a hydrogen atom, respectively; (viii) Y 3 represents a -O-(C 1 -
  • the linker-drug (or “linker-payload”) moiety -(L-D) may comprise a compounds in Table B or an enantiomer, diastereoisomer, deuterated derivative, and/or a pharmaceutically acceptable salt of any of the foregoing.
  • the BH3 mimetics D1 and/or D2 comprises a formula selected from Table A3 or A3a. d.
  • one of D1 and D2 comprises a topoisomerase 1 inhibitor, or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or addition salt thereof with a pharmaceutically acceptable acid or base.
  • D1 or D2 comprises a topoisomerase 1 inhibitor linked to the dual linker, wherein the topoisomerase 1 inhibitor is topotecan, exatecan, deruxtecan or SN-38.
  • D 1 or D 2 comprises a formula selected from Table A4 or A4a. e.
  • one of D1 and D2 comprises an anti-mitotic drug, or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or addition salt thereof with a pharmaceutically acceptable acid or base.
  • one of D1 and D2 comprises an anti-mitotic drug linked to the dual linker, wherein the anti-mitotic drug is monomethyl auristatin E (MMAE) or a taxane.
  • MMAE monomethyl auristatin E
  • the taxane is selected from docetaxel, paclitaxel, or cabazitaxel.
  • Drug loading is represented by p (or 2a in ADCs of formula (1) of the present disclosure), and is also referred to herein as the drug-to-antibody ratio (DAR). Drug loading may range from 2 to 32 drug moieties per antibody or antigen-binding fragment.
  • a is an integer from 1 to 16.
  • a is an integer from 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • a is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.
  • a is an integer from 1 to 16. In some embodiments, a is an integer from 1 to 8. In some embodiments, a is an integer from 1 to 5. In some embodiments, a is an integer from 2 to 4. In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, a is 2. In some embodiments, a is 4. [357] Drug loading may be limited by the number of attachment sites on the antibody or antigen- binding fragment. In some embodiments, the linker moiety (L) of the ADC attaches to the antibody or antigen-binding fragment through a chemically active group on one or more amino acid residues on the antibody or antigen-binding fragment.
  • the linker may be attached to the antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C- terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues).
  • a free amino, imino, hydroxyl, thiol, or carboxyl group e.g., to the N- or C- terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues.
  • the site to which the linker is attached can be a natural residue in the amino acid sequence of the antibody or antigen-binding fragment, or it can be introduced into the antibody or antigen-binding fragment, e.g., by DNA recombinant technology (e.g., by introducing a cysteine residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).
  • the number of drug moieties that can be conjugated to an antibody or antigen-binding fragment is limited by the number of free cysteine residues.
  • an antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached.
  • antibodies do not contain many free and reactive cysteine thiol groups that may be linked to a drug moiety. Indeed, most cysteine thiol residues in antibodies are involved in either interchain or intrachain disulfide bonds. Conjugation to cysteines can therefore, in some embodiments, require at least partial reduction of the antibody. Over-attachment of linker-toxin to an antibody may destabilize the antibody by reducing the cysteine residues available to form disulfide bonds.
  • an optimal drug:antibody ratio should increase potency of the ADC (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody or antigen-binding fragment.
  • an optimal ratio may be 2, 4, 6, or 8.
  • an optimal ratio may be 2 or 4.
  • an antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation in order to generate one or more free cysteine residues.
  • An antibody in some embodiments, may be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2- carboxyethyl)phosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tris(2- carboxyethyl)phosphine
  • Unpaired cysteines may be generated through partial reduction with limited molar equivalents of TCEP, which can reduce the interchain disulfide bonds which link the light chain and heavy chain (one pair per H-L pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgG1) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol.1045:145-71).
  • disulfide bonds within the antibodies are reduced electrochemically, e.g., by employing a working electrode that applies an alternating reducing and oxidizing voltage.
  • This approach can allow for on-line coupling of disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g., US 2014/0069822)).
  • an analytical device e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer
  • a chemical separation device e.g., a liquid chromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g., US 2014/0069822)
  • an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as cysteine.
  • the drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limiting reductive conditions for cysteine thiol modification; and/or (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker- drug attachments.
  • free cysteine residues are introduced into the amino acid sequence of the antibody or antigen-binding fragment.
  • cysteine engineered antibodies can be prepared wherein one or more amino acids of a parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e. mutated.
  • a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a "ThioFab.”
  • a parent monoclonal antibody may be engineered to form a "ThioMab.”
  • a single site mutation yields a single engineered cysteine residue in a ThioFab, whereas a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody.
  • DNA encoding an amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, e.g., the methods described in WO 2006/034488). These methods include, but are not limited to, preparation by site- directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides.
  • ADCs of Formula (1) include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al.
  • one or more free cysteine residues are already present in an antibody or antigen-binding fragment, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody or antigen-binding fragment to a drug moiety.
  • the resulting product can be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody or antigen-binding fragment in the mixture.
  • the drug loading in a mixture of ADCs resulting from a conjugation reaction ranges from 1 to 16 drug moieties attached per antibody or antigen- binding fragment.
  • the average number of drug moieties per antibody or antigen-binding fragment i.e., the average drug loading, or average p
  • the average number of drug moieties per antibody or antigen-binding fragment may be calculated by any conventional method known in the art, e.g., by mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and/or high- performance liquid chromatography (e.g., HIC-HPLC).
  • the average number of drug moieties per antibody or antigen-binding fragment is determined by liquid chromatography-mass spectrometry (LC-MS).
  • the average number of drug moieties per antibody or antigen-binding fragment is from about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is from about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8. [363] In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2.
  • the average number of drug moieties per antibody or antigen- binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2. [364] In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen- binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5.
  • the average number of drug moieties per antibody or antigen-binding fragment is 4. [365] In some embodiments, the term “about,” as used with respect to the average number of drug moieties per antibody or antigen-binding fragment, means plus or minus 20%, 15%, 10%, 5%, or 1%. In one embodiment, the term “about” refers to a range of values which are 10% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 1% more or less than the specified value.
  • ADC compounds may be identified in the mixture by mass spectroscopy and separated by, e.g., UPLC or HPLC, e.g. hydrophobic interaction chromatography (HIC-HPLC).
  • UPLC or HPLC e.g. hydrophobic interaction chromatography
  • a homogeneous or nearly homogenous ADC product with a single loading value may be isolated from the conjugation mixture, e.g., by electrophoresis or chromatography.
  • higher drug loading e.g., p > 16
  • the drug loading for an ADC of the present disclosure ranges from about 2 to about 16, about 2 to about 10, about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.
  • a drug loading and/or an average drug loading of about 2 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and provides beneficial properties.
  • a drug loading and/or an average drug loading of about 4 or about 6 or about 8 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and provides beneficial properties.
  • a drug loading and/or an average drug loading of less than about 2 may result in an unacceptably high level of unconjugated antibody species, which can compete with the ADC for binding to a target antigen and/or provide for reduced treatment efficacy.
  • a drug loading and/or average drug loading of more than about 16 may result in an unacceptably high level of product heterogeneity and/or ADC aggregation.
  • the ADCs comprise an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a drug moiety (e.g., a BH3 mimetic), and a linker that joins the drug moiety and the antibody or antigen-binding fragment.
  • the ADCs can be prepared using a linker having reactive functionalities for covalently attaching to the drug moiety and to the antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment is functionalized to prepare a functional group that is reactive with a linker or a drug-linker intermediate.
  • a cysteine thiol of an antibody or antigen-binding fragment can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.
  • an antibody or antigen- binding fragment is prepared with bacterial transglutaminase (BTG) - reactive glutamines specifically functionalized with an amine containing cyclooctyne BCN (N-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9- ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane) moiety.
  • BCG transglutaminase
  • BCN cyclooctyne
  • site-specific conjugation of a linker or a drug-linker intermediate to a BCN moiety of an antibody or antigen-binding fragment is performed, e.g., as described and exemplified herein.
  • an ADC is produced by contacting an antibody or antigen-binding fragment with a linker and a drug moiety (e.g., a BH3 mimetic) in a sequential manner, such that the antibody or antigen-binding fragment is covalently linked to the linker first, and then the pre-formed antibody-linker intermediate reacts with the drug moiety.
  • a linker and a drug moiety e.g., a BH3 mimetic
  • an ADC is produced by contacting an antibody or antigen-binding fragment with a linker-drug compound pre- formed by reacting a linker with a drug moiety.
  • the pre-formed linker-drug compound may or may not be subjected to a purification step prior to contacting the antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment contacts the linker and the drug moiety in one reaction mixture, allowing simultaneous formation of the covalent bonds between the antibody or antigen- binding fragment and the linker, and between the linker and the drug moiety.
  • This method of producing ADCs may include a reaction, wherein the antibody or antigen-binding fragment contacts the antibody or antigen-binding fragment prior to the addition of the linker to the reaction mixture, and vice versa.
  • an ADC is produced by reacting an antibody or antigen-binding fragment with a linker joined to a drug moiety, such as a BH3 mimetic, under conditions that allow conjugation.
  • the ADCs prepared according to the methods described above may be subjected to a purification step.
  • the purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof.
  • compositions described herein e.g., the disclosed ADC compounds and compositions, in treating a subject for a disorder, e.g., a cancer.
  • compositions e.g., ADCs
  • ADCs may be administered alone or in combination with at least one additional inactive and/or active agent, e.g., at least one additional therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and dosing regimen.
  • Treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accordingly.
  • Efficacy measures include, but are not limited to, a cytostatic and/or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and/or prolonged survival. [373] Methods of determining whether an ADC exerts a cytostatic and/or cytotoxic effect on a cell are known.
  • the cytotoxic or cytostatic activity of an ADC can be measured by, e.g., exposing mammalian cells expressing a target antigen of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 6 days; and measuring cell viability (e.g., using a CellTiter-Glo® (CTG) or MTT cell viability assay).
  • CCG CellTiter-Glo®
  • MTT cell viability assay Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.
  • Necrosis is typically accompanied by increased permeability of the plasma membrane, swelling of the cell, and rupture of the plasma membrane.
  • Apoptosis can be quantitated, for example, by measuring DNA fragmentation.
  • Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) 2:34-7 (Roche Molecular Biochemicals).
  • Apoptosis may also be determined by measuring morphological changes in a cell.
  • loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide).
  • a fluorescent dye such as, for example, acridine orange or ethidium bromide.
  • a method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp.3.17.1-3.17.16).
  • Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane.
  • a DNA dye e.g., acridine orange, ethidium bromide, or propidium iodide
  • Apoptosis may also be determined, in some embodiments, by screening for caspase activity.
  • a Caspase-Glo® Assay can be used to measure activity of caspase-3 and caspase-7.
  • the assay provides a luminogenic caspase-3/7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis.
  • adding Caspase-Glo® 3/7 Reagent in an “add-mix-measure” format may result in cell lysis, followed by caspase cleavage of the substrate and generation of a “glow-type” luminescent signal, produced by luciferase.
  • luminescence may be proportional to the amount of caspase activity present, and can serve as an indicator of apoptosis.
  • Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.
  • Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMARTM blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473- 6).
  • the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.
  • Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolically active cells.
  • in vitro potency and/or cell viability of prepared ADCs or antineoplastic payloads such as BH3 mimetic compounds (e.g., MCl-1 inhibitor, Bcl-xL inhibitor or Bcl- 2 inhibitor) or topoisomerase 1 inhibitors (e.g., topotecan, exatecan, deruxtecan or SN-38) or anti-mitotic drugs (e.g., monomethyl auristatin E (MMAE) or a taxane) may be assessed using a CellTiter-Glo® (CTG) cell viability assay, as described in the examples provided herein.
  • CCG CellTiter-Glo®
  • the single reagent (CellTiter-Glo® Reagent) is added directly to cells cultured in serum- supplemented medium.
  • the addition of reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • Cell viability may also be measured, e.g., by measuring the reduction of tetrazolium salts.
  • in vitro potency and/or cell viability of prepared ADCs or antineoplastic payloads such as BH3 mimetic compounds (e.g., MCl-1 inhibitor, Bcl-xL inhibitor or Bcl-2 inhibitor) or topoisomerase 1 inhibitors (e.g., topotecan, exatecan, deruxtecan or SN-38) or anti-mitotic drugs (e.g., monomethyl auristatin E (MMAE) or a taxane) may be assessed using an MTT cell viability assay, as described in the examples provided herein.
  • BH3 mimetic compounds e.g., MCl-1 inhibitor, Bcl-xL inhibitor or Bcl-2 inhibitor
  • topoisomerase 1 inhibitors e.g., topotecan, exatecan, deruxtecan or SN-38
  • anti-mitotic drugs e.g., monomethyl auristatin E (MMAE) or a taxane
  • the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH.
  • the resulting intracellular purple formazan can then be solubilized and quantified by spectrophotometric means.
  • the present disclosure features a method of killing, inhibiting or modulating the growth of a cancer cell or tissue by disrupting the expression and/or activity of Bcl-2 family protein (e.g., Mcl-1, Bcl-2 and/or Bcl-xL) and/or one or more upstream modulators or downstream targets thereof.
  • Bcl-2 family protein e.g., Mcl-1, Bcl-2 and/or Bcl-xL
  • the method may be used with any subject where disruption of Bcl-2 family protein expression and/or activity provides a therapeutic benefit.
  • Subjects that may benefit from disrupting Bcl-2 family protein expression and/or activity include, but are not limited to, those having or at risk of having a cancer such as a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • the disclosed ADCs may be administered in any cell or tissue that expresses CD7, such as a CD7-expressing cancer cell or tissue.
  • An exemplary embodiment includes a method of killing a CD7-expressing cancer cell or tissue. The method may be used with any cell or tissue that expresses CD7, such as a cancerous cell or a metastatic lesion.
  • Non-limiting examples of CD7- expressing cancers include , multiple myeloma, plasma cell myeloma, leukemia, lymphoma, acute myeloid leukemia, , bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, myelogenous leukemia, myeloma, chronic lymphocytic leukemia, or spleen cancer.
  • Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., an amount sufficient to kill the cell.
  • the method can be used on cells in culture, e.g., in vitro, in vivo, ex vivo, or in situ.
  • cells that express CD7 e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells
  • the contacting step can be affected by adding the ADC to the culture medium.
  • the method will result in killing of cells expressing CD7, including in particular cancer cells expressing CD7.
  • the ADC can be administered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to have an effect in vivo.
  • the in vivo effect of a disclosed ADC therapeutic composition can be evaluated in a suitable animal model.
  • xenogeneic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med.3:402-8). Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
  • In vivo assays that evaluate the promotion of tumor death by mechanisms such as apoptosis may also be used.
  • xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
  • a disorder e.g., a cancer.
  • the compositions described herein, e.g., the ADCs disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes.
  • the therapeutic methods include administering to a subject having or suspected of having a cancer a therapeutically effective amount of a composition comprising an ADC with a targeting antibody that binds to an antigen (e.g., CD7) (1) expressed on a cancer cell, (2) is accessible to binding, and/or (3) is localized or predominantly expressed on a cancer cell surface as compared to a non-cancer cell.
  • An exemplary embodiment is a method of treating a subject having or suspected of having a cancer, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein, e.g., an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein).
  • the cancer expresses a target antigen CD7.
  • the cancer is a tumor or a hematological cancer.
  • the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, sarcoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.
  • the cancer is a lymphoma or gastric cancer.
  • Another exemplary embodiment is a method of delivering antineoplastic payloads (e.g. BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs) to a cell expressing CD7, comprising conjugating the antineoplastic payloads (e.g. BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs) to an antibody that immunospecifically binds to a CD7 epitope and exposing the cell to the ADC.
  • antineoplastic payloads e.g. BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs
  • Exemplary cancer cells that express CD7 for which the ADCs of the present disclosure are indicated include hematological cancer cells, such as multiple myeloma, plasma cell myeloma, leukemia, lymphoma, , acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, , myelogenous leukemia, myeloma, chronic lymphocytic leukemia.
  • hematological cancer cells such as multiple myeloma, plasma cell myeloma, leukemia, lymphoma, , acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, , myelogenous leukemia, myel
  • the present disclosure further provides methods of reducing or inhibiting growth of a tumor (e.g., a CD7-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC.
  • a tumor e.g., a CD7-expressing tumor
  • the treatment is sufficient to reduce or inhibit the growth of the patient's tumor, reduce the number or size of metastatic lesions, reduce tumor load, reduce primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or improve the quality of life.
  • the tumor is resistant or refractory to treatment with the antibody or antigen-binding fragment of the ADC (e.g., anti-CD7 antibody or antigen- binding fragment) when administered alone, and/or the tumor is resistant or refractory to treatment with a BH3 mimetic drug moiety when administered alone.
  • An exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein).
  • the tumor expresses a target antigen CD7.
  • the tumor is a multiple myeloma, plasma cell myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, myelogenous leukemia, myeloma, chronic lymphocytic leukemia, or spleen cancer
  • administration of the ADC, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to growth in the absence of treatment.
  • An exemplary embodiment is a method of reducing or inhibiting the growth of a hematological cancer in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein).
  • the hematological cancer expresses a target antigen.
  • the target antigen is CD7.
  • the hematological cancer is chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non- Hodgkin's lymphoma or myelodysplasia syndrome (MDS).
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • CML chronic myelomonocytic leukemia
  • AoL acute monocytic leukemia
  • administration of the ADC, composition, or pharmaceutical composition reduces or inhibits the growth of the hematological cancer by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to growth in the absence of treatment.
  • Another exemplary embodiment is a method of delaying or slowing the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein).
  • the tumor expresses a target antigen CD7.
  • the tumor is multiple myeloma, plasma cell myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, myelogenous leukemia, myeloma, chronic lymphocytic leukemia, or spleen cancer.
  • administration of the ADC, composition, or pharmaceutical composition delays or slows the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to growth in the absence of treatment.
  • the present disclosure further provides methods of reducing or slowing the expansion of a cancer cell population (e.g., a CD7-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC.
  • An exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein).
  • the cancer cell population expresses a target antigen CD7.
  • the cancer cell population is from a tumor or a hematological cancer.
  • the cancer cell population is from a multiple myeloma, plasma cell myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma,spleen cancer.
  • the cancer cell population is from a lymphoma.
  • administration of the ADC, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to the population in the absence of treatment.
  • administration of the ADC, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to expansion in the absence of treatment.
  • An exemplary embodiment is a method of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) by providing a biological sample from the subject; contacting the sample with the ADC; and detecting binding of the ADC to cancer cells in the sample.
  • the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.
  • the method comprises providing a biological sample from the subject; contacting the sample with the ADC; and detecting one or more markers of cancer cell death in the sample (e.g., increased expression of one or more apoptotic markers, reduced expansion of a cancer cell population in culture, etc.).
  • markers of cancer cell death in the sample e.g., increased expression of one or more apoptotic markers, reduced expansion of a cancer cell population in culture, etc.
  • An exemplary embodiment is an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) for use in treating a subject having or suspected of having a cancer (e.g., a CD7-expressing cancer).
  • Another exemplary embodiment is a use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) in treating a subject having or suspected of having a cancer (e.g., a CD7-expressing cancer).
  • Another exemplary embodiment is a use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) in a method of manufacturing a medicament for treating a subject having or suspected of having a cancer (e.g., aa CD7-expressing cancer).
  • ADCs of the present disclosure may be administered to a non-human mammal expressing an antigen with which the ADC is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (e.g., testing of dosages and time courses of administration).
  • the therapeutic compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier suitable for the desired delivery method.
  • An exemplary embodiment is a pharmaceutical composition
  • a pharmaceutical composition comprising an ADC of the present disclosure and a pharmaceutically acceptable carrier, e.g., one suitable for a chosen means of administration, e.g., intravenous administration.
  • the pharmaceutical composition may also comprise one or more additional inactive and/or therapeutic agents that are suitable for treating or preventing, for example, a cancer (e.g., a standard-of-care agent, etc.).
  • the pharmaceutical composition may also comprise one or more carrier, excipient, and/or stabilizer components, and the like. Methods of formulating such pharmaceutical compositions and suitable formulations are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA).
  • Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof.
  • isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.
  • a pharmaceutical composition of the present disclosure can be administered by a variety of methods known in the art. The route and/or mode of administration may vary depending upon the desired results. In some embodiments, the therapeutic formulation is solubilized and administered via any route capable of delivering the therapeutic composition to the cancer site.
  • routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous), intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like.
  • the administration is intravenous, subcutaneous, intraperitoneal, or intramuscular.
  • the pharmaceutically acceptable carrier should be suitable for the route of administration, e.g., intravenous or subcutaneous administration (e.g., by injection or infusion).
  • the active compound(s) i.e., the ADC and/or any additional therapeutic agent, may be coated in a material to protect the compound(s) from the action of acids and other natural conditions that may inactivate the compound(s).
  • the therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage, and may be in a variety of forms. These include, for example, liquid, semi- solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the intended mode of administration and therapeutic application.
  • the disclosed ADCs can be incorporated into a pharmaceutical composition suitable for parenteral administration.
  • the injectable solution may be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule, or pre-filled syringe, or other known delivery or storage device.
  • one or more of the ADCs or pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject.
  • a therapeutically effective amount or efficacious amount of a disclosed composition e.g., a disclosed ADC, is employed in the pharmaceutical compositions of the present disclosure.
  • compositions e.g., one comprising an ADC
  • the composition may be formulated into a pharmaceutically acceptable dosage form by conventional methods known in the art. Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art. [402] Dosage regimens for compositions disclosed herein, e.g., those comprising ADCs alone or in combination with at least one additional inactive and/or active therapeutic agent, may be adjusted to provide the optimum desired response (e.g., a therapeutic response).
  • a single bolus of one or both agents may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose of one or both agents may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation.
  • treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration.
  • the ADC is administered to the patient daily, weekly, monthly, or any time period in between.
  • specific dosage regimens may be adjusted over time according to the individual’s need, and the professional judgment of the treating clinician.
  • Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Dosage values for compositions comprising an ADC and/or any additional therapeutic agent(s) may be selected based on the unique characteristics of the active compound(s), and the particular therapeutic effect to be achieved. A physician or veterinarian can start doses of the ADC employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • effective doses of the compositions of the present disclosure, for the treatment of a cancer may vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the selected dosage level may also depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt, or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • Treatment dosages may be titrated to optimize safety and efficacy.
  • Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or in animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index.
  • the data obtained from in vitro and in vivo assays can be used in estimating or formulating a range of dosage for use in humans.
  • the compositions and methods disclosed herein may initially be evaluated in xenogeneic cancer models (e.g., an NCI-H929 multiple myeloma mouse model).
  • an ADC or composition comprising an ADC is administered on a single occasion. In other embodiments, an ADC or composition comprising an ADC is administered on multiple occasions. Intervals between single dosages can be, e.g., daily, weekly, monthly, or yearly. Intervals can also be irregular, based on measuring blood levels of the administered agent (e.g., the ADC) in the patient in order to maintain a relatively consistent plasma concentration of the agent.
  • the dosage and frequency of administration of an ADC or composition comprising an ADC may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
  • a relatively higher dosage at relatively shorter intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of one or more symptoms of disease. Thereafter, the patient may be administered a lower, e.g., prophylactic regime.
  • the above therapeutic approaches can be combined with any one of a wide variety of additional surgical, chemotherapy, or radiation therapy regimens.
  • the ADCs or compositions disclosed herein are co-formulated and/or co-administered with one or more additional therapeutic agents, e.g., one or more chemotherapeutic agents, one or more standard-of-care agents for the particular condition being treated.
  • Kits for use in the therapeutic and/or diagnostic applications described herein are also provided.
  • Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein.
  • a label may be present on or with the container(s) to indicate that an ADC or composition within the kit is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application.
  • a label may also indicate directions for either in vivo or in vitro use, such as those described herein.
  • a kit comprises an ADC or composition comprising an ADC.
  • the kit further comprises one or more additional components, including but not limited to: instructions for use; other reagents, e.g., a therapeutic agent (e.g., a standard-of-care agent); devices, containers, or other materials for preparing the ADC for administration; pharmaceutically acceptable carriers; and devices, containers, or other materials for administering the ADC to a subject.
  • a therapeutic agent e.g., a standard-of-care agent
  • kits, or other materials for preparing the ADC for administration e.g., a standard-of-care agent
  • pharmaceutically acceptable carriers e.g., a standard-of-care agent
  • the kit comprises an ADC and instructions for use of the ADC in treating, preventing, and/or diagnosing a cancer.
  • BCL2 family member e.g., Bcl-xL
  • Antibody-drug conjugates that may not be sufficiently effective as monotherapy to treat cancer can be administered in combination with other therapeutic agents (including non-targeted and targeted therapeutic agents) or radiation therapy (including radioligand therapy) to provide therapeutic benefit.
  • other therapeutic agents including non-targeted and targeted therapeutic agents
  • radiation therapy including radioligand therapy
  • the ADCs described herein sensitize tumor cells to the treatment with other therapeutic agents (including standard of care chemotherapeutic agents to which the tumor cells may have developed resistance) and/or radiation therapy.
  • antibody drug conjugates described herein are administered to a subject having cancer in an amount effective to sensitize the tumor cells.
  • the term “sensitize” means that the treatment with ADC increases the potency or efficacy of the treatment with other therapeutic agents and/or radiation therapy against tumor cells. 3.
  • Combination Therapies [410] In some embodiments, the present disclosure provides methods of treatment wherein the antibody-drug conjugates disclosed herein are administered in combination with one or more (e.g., 1 or 2) additional therapeutic agents. Exemplary combination partners are disclosed herein. [411] In some embodiments, a combination described herein comprises a vinca alkaloid. [412] Vinca alkaloids include, but are not limited to, vincristine, vinblastine, and leurosine. [413] In some embodiments, a combination described herein comprises a topoisomerase inhibitor.
  • Topoisomerase inhibitors include, but are not limited to, topotecan, irinotecan, camptothecin, diflomotecan, lamellarin D, ellipticines, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, aurintricarboxylic acid, and HU-331.
  • a combination described herein comprises a hypomethylating agent (HMA).
  • the HMA is chosen from decitabine or azacitidine.
  • a combination described herein comprises a glucocorticoid.
  • the glucocorticoid is dexamethasone.
  • a combination described herein comprises asparaginase.
  • a combination described herein comprises an inhibitor acting on any pro- survival proteins of the Bcl2 family including Bcl2i, Bclxli and Mcl1i inhibitor.
  • a combination described herein comprises a Bcl-2 inhibitor.
  • the Bcl-2 inhibitor is venetoclax (also known as ABT-199): ( venetoclax).
  • the Bcl-2 inhibitor is selected from the compounds described in WO 2013/110890 and WO 2015/011400.
  • the Bcl-2 inhibitor comprises navitoclax (ABT-263), ABT-737, BP1002, SPC2996, APG-1252, obatoclax mesylate (GX15-070MS), PNT2258, Zn-d5, BGB-11417, or oblimersen (G3139).
  • the Bcl-2 inhibitor is N-(4- hydroxyphenyl)-3-[6-[(3S)-3-(morpholinomethyl)-3,4-dihydro-1H-isoquinoline-2-carbonyl]-1,3- benzodioxol-5-yl]-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide, compound A1:
  • the Bcl-2 inhibitor is (S)-5-(5-chloro-2-(3-(morpholinomethyl)-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)phenyl)-N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4- hydroxyphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxamide), compound A2: (compound A2).
  • the antibody-drug conjugates or combinations disclosed herein are suitable for the treatment of cancer in vivo.
  • the combination can be used to inhibit the growth of cancerous tumors.
  • the combination can also be used in combination with one or more of: a standard of care treatment (e.g., for cancers or infectious disorders), a vaccine (e.g., a therapeutic cancer vaccine), a cell therapy, a hormone therapy (e.g., with anti-estrogens or anti-androgens), a radiation therapy, surgery, or any other therapeutic agent or modality, to treat a disorder herein.
  • a standard of care treatment e.g., for cancers or infectious disorders
  • a vaccine e.g., a therapeutic cancer vaccine
  • a cell therapy e.g., a hormone therapy (e.g., with anti-estrogens or anti-androgens)
  • a radiation therapy e.g., surgery, or any other therapeutic agent or modality
  • the combination can be administered together with an antigen of interest.
  • a combination disclosed herein can be administered in either order or simultaneously.
  • the present invention provides various methods of conjugating Linker-Drug groups of the invention to antibodies or antibody fragments to produce Antibody Drug Conjugates which comprise a linker having one or more hydrophilic moieties.
  • a general reaction scheme for the formation of Antibody Drug Conjugates of Formula (A) is shown in Scheme 1 below: Scheme 1 [424] where: RG2 is a reactive group which reacts with a compatible R100 group to form a corresponding R 1 group (such groups are illustrated in Table F and Table G). D 1 , D 2 , R 1 , L 1 , L 2’ , L 3’ , Ab, W, a, R 1 and R 100 are as defined herein.
  • Scheme 3 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab4) wherein a free thiol group generated from the engineered cysteine residues in the antibody react with an R 100 group (where R 100 is a maleimide) to covalently attach the Linker-Drug group to the antibody via an R 1 group (where R 1 is a succinimide ring).
  • R 100 where R 100 is a maleimide
  • R 1 where R 1 is a succinimide ring
  • Scheme 3 shows the antibody having four free thiol groups.
  • Linker-Drug groups are conjugated to antibodies via lysine residues in the antibodies.
  • Scheme 4 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab6) wherein a free amine group from the lysine residues in the antibody react with an R 100 group (where R 100 is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl) to covalently attach the Linker- Drug group to the antibody via an R 1 group (where R 1 is an amide).
  • R 100 is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl
  • R 1 group where R 1 is an amide
  • Linker-Drug groups are conjugated to antibodies via formation of an oxime bridge at the naturally occurring disulfide bridges of an antibody.
  • the oxime bridge is formed by initially creating a ketone bridge by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1,3-dihaloacetone (e.g.1,3-dichloroacetone). Subsequent reaction with a Linker-Drug group comprising a hydroxyl amine thereby form an oxime linkage (oxime bridge) which attaches the Linker- Drug group to the antibody (see for example WO2014/083505).
  • Scheme 5 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab9).
  • RG2 is a reactive group which reacts with a compatible R100 group to form a corresponding R 1 group (such groups are illustrated in Table F and Table G).
  • D 1 , D 2 , R 1 , L 1 , W, L 2 , L 3 , E 1 , E 2 , L 4 , L 5 , A 1 , A 2 , R 2 , R 3 , Ab, a and R 100 are as defined herein.
  • Linker-Drug groups are conjugated to antibodies via modified cysteine residues in the antibodies (see for example WO2014/124316).
  • Scheme 8 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab13) wherein a free thiol group generated from the engineered cysteine residues in the antibody react with an R 100 group (where R 1 is a maleimide) to covalently attach the Linker-Drug group to the antibody via an R 1 group (where R 1 is a succinimide ring).
  • R 100 group where R 1 is a maleimide
  • R 1 group where R 1 is a succinimide ring
  • Linker-Drug groups are conjugated to antibodies via lysine residues in the antibodies.
  • Scheme 9 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab15) wherein a free amine group from the lysine residues in the antibody react with an R 100 group (where R 100 is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl) to covalently attach the Linker- Drug group to the antibody via an R 1 group (where R 1 is an amide).
  • R 100 group where R 100 is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl
  • R 1 group where R 1 is an amide
  • Linker-Drug groups are conjugated to antibodies via formation of an oxime bridge at the naturally occurring disulfide bridges of an antibody.
  • the oxime bridge is formed by initially creating a ketone bridge by reduction of an interchain disulfide bridge of the antibody and re-bridging using a 1,3-dihaloacetone (e.g.1,3-dichloroacetone). Subsequent reaction with a Linker-Drug group comprising a hydroxyl amine thereby form an oxime linkage (oxime bridge) which attaches the Linker- Drug group to the antibody (see for example WO2014/083505).
  • Scheme 10 illustrates this approach for the formation of Antibody Drug Conjugates of Formula (Ab18).
  • conjugates have favorable properties, for example properties that would make them easier to manufacture, easier to administer to patients, more efficacious, and/or potentially safer for patients.
  • One example is the determination of molecular size by size exclusion chromatography (SEC) wherein the amount of desired antibody species in a sample is determined relative to the amount of high molecular weight contaminants (e.g., dimer, multimer, or aggregated antibody) or low molecular weight contaminants (e.g., antibody fragments, degradation products, or individual antibody chains) present in the sample.
  • SEC size exclusion chromatography
  • hydrophobicity by hydrophobic interaction chromatography (HIC) wherein the hydrophobicity of a sample is assessed relative to a set of standard antibodies of known properties.
  • HIC hydrophobic interaction chromatography
  • Payload P1 was prepared according to the method described in Example 30 of International PCT publication WO2015/097123. Below is a table showing structures of exemplary payloads P1, P2, and P4- P6.
  • payloads P1, P2, and P4-P6 are BH3 mimetics, which correspond to Mcl-1 inhibitor D1-1, Bcl-2 inhibitors D3-1, D3-3, and Bcl-xL inhibitors D2-25 and D2-1, respectively.
  • TLC Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60 F254 silica-gel.
  • Microwave Reactions [447] Microwave heating was performed with a CEM Discover® SP, or with an Anton Paar Monowave Microwave Reactor.
  • NMR [449] 1H-NMR measurements were performed on a Bruker Avance III 500 MHz spectrometer, a Bruker Avance III 400 MHz spectrometer, or a Bruker DPX-400 spectrometer using DMSO-d 6 or CDCl 3 as solvent.
  • 1H NMR data is in the form of delta values, given in part per million (ppm), using the residual peak of the solvent (2.50 ppm for DMSO-d 6 and 7.26 ppm for CDCl 3 ) as internal standard.
  • Splitting patterns are designated as: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sept (septet), m (multiplet), br s (broad singlet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), ddd (doublet of doublet of doublets).
  • Acidic LCMS KINATEX XB-C18-100A, 2.6 !m, 50 mm*2.1 mm column at 40 °C, at a flow rate of 1 mL min-1 using 0.02% v/v aqueous formic acid (Solvent A) and 0.02% v/v formic acid in acetonitrile (Solvent B) with a gradient starting from 100% Solvent A and finishing at 100% Solvent B over various/certain duration of time.
  • Certain other compounds of the present invention were characterized HPLC-MS under specific named methods as follows. For all of these methods UV detection was by diode array detector at 230, 254, and 270 nm. Sample injection volume was 1 !L.
  • LCMS-V-B methods [459] Using an Agilent 1200 SL series instrument linked to an Agilent MSD 6140 single quadrupole with an ESI-APCI multimode source (Methods LCMS-V-B1 and LCMS-V-B2) or using an Agilent 1290 Infinity II series instrument connected to an Agilent TOF 6230 with an ESI-jet stream source (Method LCMS-V-B1); column: Thermo Accucore 2.6 ⁇ m, C18, 50 mm x 2.1 mm at 55 oC.
  • the mass spectrometer was a Waters Micromass ZQ2000 spectrometer, operating in positive or negative ion electrospray ionisation modes, with a molecular weight scan range of 150 to 1000.
  • HPLC-V-B methods [472] Performed on an AccQPrep HP125 (Teledyne ISCO) system, with a Gemini® NX 5 ⁇ m C18(2), 150 mm ⁇ 21.2 mm i.d. column from Phenomenex, running at a flow rate of 20 cm 3 min -1 with UV (214 and 254 nm) and ELS detection. [473] Method HPLC-V-B1 (pH 4): [474] Solvent A: water + 0.08% (v/v) formic acid; solvent B: acetonitrile + 0.08% (v/v) formic acid.
  • High-resolution MS [482] High-resolution mass spectra were acquired on an Agilent 6230 time-of-flight mass spectrometer P[_TZZPO aT ⁇ S L @P ⁇ G ⁇ PLW PVPN ⁇ Y]Z ⁇ Lc TYX ]Y_ ⁇ NP TX ZY]T ⁇ T ⁇ P TYX WYOP( ?XUPN ⁇ TYX] YQ *(/nV aP ⁇ P OT ⁇ PN ⁇ PO to the mass spectrometer at a flow rate 1.5 ml/min (5mM ammonium-formate in water and acetonitrile gradient program), using an Agilent 1290 Infinity HPLC system.
  • Jet Stream parameters drying gas (N2) flow and temperature: 8.0 l/min and 325 °C, respectively; nebulizer gas (N2) pressure: 30 psi; capillary voltage: 3000 V; sheath gas flow and temperature: 325 °C and 10.0 l/min; TOFMS parameters: fragmentor voltage: 100 V; skimmer potential: 60 V; OCT 1 RF Vpp:750 V. Full-scan mass spectra were acquired over the m/z range 105-1700 at an acquisition rate of 995.6 ms/spectrum and processed by Agilent MassHunter B.04.00 software.
  • Step A [(pent-4-yn-1-yloxy)methyl]benzene [501] To an oven-dried flask was added 4-pentyn-1-ol (11.1 mL, 119 mmol, 1 eq) in THF (100 mL) and the solution was cooled to 0°C.
  • Step B [(hex-4-yn-1-yloxy)methyl]benzene [503] To an oven-dried flask was added the product from Step A (19.5 g, 112 mmol, 1 eq) and tetrahydrofuran (200 mL) and the solution was cooled to -78 °C.
  • n-Butyllithium (2M solution in hexanes, 66.9 mL, 135 mmol, 1.2 eq) was added dropwise over 30 min and the reaction was stirred for 1 h then iodomethane (10.5 mL, 168 mmol, 1.5 eq) was added dropwise and the mixture was allowed to warm to 0 °C over 1 h.
  • Step C 4-[3-(benzyloxy)propyl]-3,6-dichloro-5-methylpyridazine
  • a solution of 3,6-dichloro-1,2,4,5-tetrazine (5 g, 33.1 mmol, 1 eq) and the product from Step B (7.48 g, 39.8 mmol, 1.2 eq) in tetrahydrofuran (30 mL) was heated at 160°C for 19 h in a sealed flask. The reaction was allowed to cool to ambient temperature then concentrated in vacuo.
  • Step D 3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propan-1-ol
  • Preparation B 3,6-dichloro-4-(3-iodopropyl)-5-methyl-pyridazine
  • Step A 3-bromo-5,7-dimethyladamantane-1-carboxylic acid
  • bromine 30.7 mL, 600 mmol, 5 eq
  • 3,5- dimethyladamantane-1-carboxylic acid 25 g, 1 eq
  • EtOAc the reaction mixture was treated carefully with a saturated solution of sodium-thiosulfate at 0 °C and stirred for 15 min.
  • Step B 3-bromo-5,7-dimethyl-1-adamantyl-methanol
  • Step C 1-[3-bromo-5,7-dimethyl-1-adamantyl]methyl]pyrazole [516] To the product from Step B (16.19 g, 59.26 mmol) and 1H-pyrazole (4.841 g, 1.2 eq) in toluene (178 mL) was added cyanomethylenetributylphosphorane (18.64 mL, 1.2 eq) in one portion and the reaction mixture was stirred at 90 °C for 2 h. Purification by column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (17.88 g, 93%).
  • Step D 5-methyl-1-[[-3-bromo-5,7-dimethyl-1-adamantyl]methyl]pyrazole [518] To the solution of the product from Step C (17.88 g, 55.3 mmol) in THF (277 mL) was added butyllithium (2.5 M in THF, 66 mL, 3 eq) at -78 °C, then after 1 h, iodomethane (17.2 mL, 5 eq) was added.
  • Step E 2-[[-3,5-dimethyl-7-[(5-methylpyrazol-1-yl)methyl]-1-adamantyl]oxy]ethanol
  • the mixture of the product from Step D (18.7 g, 55.3 mmol), ethylene glycol (123 mL, 40 eq), and DIPEA (48.2 mL, 5 eq) was stirred at 120 °C for 6 h. After the reaction mixture was diluted with water and extracted with EtOAc, the combined organic layers were dried and concentrated to give the desired product (18.5 g, 105%), which was used in the next step without further purification.
  • Step F tert-butyl-diphenyl-[2-[[-3,5-dimethyl-7-[(5-methylpyrazol-1-yl)methyl]-1- adamantyl]oxy]ethoxy]silane [522] To the mixture of the product from Step E (17.6 g, 55.3 mmol) and imidazole (5.65 g, 1.5 eq) in DCM (150 ml) was added tert-butyl-chloro-diphenyl-silane (18.6 g, 1.2 eq) and the reaction mixture was stirred for 1 h.
  • Step G tert-butyl-diphenyl-[2-[[3-[(4-iodo-5-methyl-pyrazol-1-yl)methyl]-5,7-dimethyl-1- adamantyl]oxy]ethoxy]silane [524] To the solution of the product from Step F (27.0 g, 48.56 mmol) in DMF (243 mL) was added N- iodosuccinimide (13.6 g, 1.25 eq) and the reaction mixture was stirred for 2 h. After the dilution with water, the mixture was extracted with DCM.
  • Step H tert-butyl-diphenyl-[2-[[3,5-dimethyl-7-[[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyrazol-1-yl]methyl]-1-adamantyl]oxy]ethoxy]silane
  • Step B methyl 3-bromo-6-(tert-butoxycarbonylamino)pyridine-2-carboxylate [531] To the product from Step A (42.7 g, 74.34 mmol) in DCM (370 mL) was added TFA (17.1 mL, 3 eq) at 0 °C and the reaction mixture was stirred for 18 h.
  • Step C methyl 3-bromo-6-[tert-butoxycarbonyl-[3-(3,6-dichloro-5-methyl-pyridazin-4- yl)propyl]amino]pyridine-2-carboxylate
  • Cs2CO3 29.5 g, 3 eq
  • Preparation B 9.9 g, 1 eq
  • Step D methyl 3-bromo-6-[3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propylamino]pyridine-2- carboxylate
  • Step C The product from Step C (17.5 g, 32.7 mmol) in 1,1,1,3,3,3-hexafluoroisopropanol (330 mL) was stirred at 110 °C for 18 h. Purification by column chromatography (silica gel, heptane and EtOAc as eluents) afforded the desired product (9.9 g, 70%).
  • Step B (4-methoxyphenyl)methyl 3-bromo-6-[3-(3,6-dichloro-5-methyl-pyridazin-4- yl)propylamino]pyridine-2-carboxylate
  • 4-methoxyphenyl)methanol (16.4 mL, 2 eq)
  • PPh 3 (34.6 g, 2 eq)
  • toluene 6 mL
  • THF 20 ml
  • Step B (4-methoxyphenyl)methyl 3-[1-[[3-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]-5,7-dimethyl- 1-adamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3- c]pyridazin-8-yl)pyridine-2-carboxylate [545] The mixture of the product from Step A (21.9 g, 21.6 mmol), Cs2CO3 (14 g, 2 eq), DIPEA (7.5 mL, 2 eq) and Pd(Ataphos) 2 Cl 2 (954 mg, 0.1 eq) in 1,4-dioxane (108 mL) was stirred at 110 °C for 18 h.
  • Step C (4-methoxyphenyl)methyl 6-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin- 8-yl)-3-[1-[[3-(2-hydroxyethoxy)-5,7-dimethyl-1-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2- carboxylate [547] To the product from Step B (8.4 g, 8.6 mmol) in THF (86 mL) was added a 1 M solution of TBAF in THF (9.4 mL, 1.1 eq) at 0 °C and the reaction mixture was stirred at room temperature for 1.5 h.
  • Step D (4-methoxyphenyl)methyl 6-[3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H- pyrido[2,3-c]pyridazin-8-yl]-3-[1-[[3-(2-hydroxyethoxy)-5,7-dimethyl-1-adamantyl]methyl]-5-methyl- pyrazol-4-yl]pyridine-2-carboxylate [549] The mixture of the product from Step C (4.7 g, 6.3 mmol), 1,3-benzothiazol-2-amine (1.9 g, 2 eq), Pd 2 dba 3 (580 mg, 0.1 eq), XantPhos (730 mg, 0.2 eq), and DIPEA (3.3 mL, 3 eq) in cyclohexanol (38 mL) was stirred at 130 °C for 2 h.
  • Step E (4-methoxyphenyl)methyl 6-[3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H- pyrido[2,3-c]pyridazin-8-yl]-3-[1-[[3,5-dimethyl-7-[2-(p-tolylsulfonyloxy)ethoxy]-1-adamantyl]methyl]- 5-methyl-pyrazol-4-yl]pyridine-2-carboxylate [551] To the product from Step D (3.83 g, 4.48 mmol) and triethylamine (1.87 mL, 3 eq) in DCM (45 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (2.19 g, 1.5 eq) and the reaction mixture was stirred for 2 h.
  • Step B methyl 2-(tert-butoxycarbonylamino)-5-(3-hydroxyprop-1-ynyl)thiazole-4-carboxylate
  • a 500 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser.
  • Step C methyl 2-(tert-butoxycarbonylamino)-5-(3-hydroxypropyl)thiazole-4-carboxylate
  • An 1 L oven-dried pressure bottle equipped with a PTFE-coated magnetic stir bar was charged with 44.75 g of the product from Step B (143.3 mmol, 1 equiv), 7.62 Pd/C ( 7.17 mmol, 0.05 equiv) in 340 mL ethanol, and then placed under a nitrogen atmosphere using hydrogenation system. After that, it was filled with 4 bar H2 gas and stirred at rt overnight. Full conversion was observed, but only the olefin product was formed.
  • Step D methyl 2-(tert-butoxycarbonylamino)-5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole-4- carboxylate
  • Step A methyl 2- ⁇ [(tert-butoxy)carbonyl][3-(3,6-dichloro-5-methylpyridazin-4- yl)propyl]amino ⁇ -5-[3-(2-fluoro-4-iodophenoxy)propyl]-1,3-thiazole-4-carboxylate
  • Step A Using Mitsunobu General Procedure I starting from 4.85 g Preparation G (9.04 mmol, 1 equiv) as the appropriate carbamate and 2 g Preparation A (9.04 mmol, 1 equiv) as the appropriate alcohol, 4.6 g of the desired product (69% Yield) was obtained.
  • Step B methyl 2-[3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propylamino]-5-[3-(2-fluoro-4-iodo- phenoxy)propyl]thiazole-4-carboxylate
  • Step B Using Deprotection with HFIP General Procedure starting from the product from Step A as the appropriate carbamate, 3.70 g the desired product (97% Yield) was obtained.
  • 1 H NMR 500 MHz, DMSO-d6$ j ZZW 1(1+ # ⁇ & + >$& 1(/3 #OO& + >$& 1(..
  • Step C methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-(2-fluoro- 4-iodo-phenoxy)propyl]thiazole-4-carboxylate
  • a suspension of 3 g of the product from Step B (4.69 mmol, 1 eq) and 1.81 g cesium carbonate (9.3853 mmol, 2 eq.) were stirred at 80°C for 3 h in 25 mL dry 1,4-dioxane to reach complete conversion.
  • Step B methyl 2-[3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3- c]pyridazin-8-yl]-5-[3-[4-[3-[tert-butyl(dimethyl)silyl]oxyprop-1-ynyl]-2-fluoro- phenoxy]propyl]thiazole-4-carboxylate [573] Using Buchwald General Procedure II starting from 2.8 g of the product from Step A (4.34 mmol, 1.0 eq.) and 1.30 g 1,3-benzothiazol-2-amine (8.67 mmol, 2.0 eq.), 2.1 g of the desired product (64% Yield) was obtained.
  • Step C methyl 2-[3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3- c]pyridazin-8-yl]-5-[3-[2-fluoro-4-(3-hydroxyprop-1-ynyl)phenoxy]propyl] thiazole-4-carboxylate [575] A 100 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser.
  • Step A (3R)-3-methyl-1,2,3,4-tetrahydroisoquinoline hydrochloride
  • Step B benzyl 6-[4-(ethoxycarbonyl)-1,5-dimethylpyrrol-2-yl]-7- ⁇ [(3R)-3-methyl-3,4-dihydro- 1H-isoquinolin-2-yl]carbonyl ⁇ -3,4-dihydro-1H-isoquinoline-2-carboxylate
  • the mixture was diluted with water (120 mL), stirred for 15 mins and the resultant cream precipitate was collected by filtration, washing with water.
  • the filter cake was dissolved in DCM and washed with sat. aq. NaHCO 3 solution and water, dried over MgSO 4 and concentrated in vacuo to afford the title product (3.7 g, 6.11 mmol, 97%).
  • Step C ethyl 1,2-dimethyl-5-(7- ⁇ [(3R)-3-methyl-3,4-dihydro-1H-isoquinolin-2-yl]carbonyl ⁇ - 1,2,3,4-tetrahydroisoquinolin-6-yl)pyrrole-3-carboxylate
  • MeOH 70 mL
  • EtOH 20 mL
  • Pd/C 100 mg
  • Step D tert-butyl 6-[4-(ethoxycarbonyl)-1,5-dimethylpyrrol-2-yl]-7- ⁇ [(3R)-3-methyl-3,4-dihydro- 1H-isoquinolin-2-yl]carbonyl ⁇ -3,4-dihydro-1H-isoquinoline-2-carboxylate [614] To a solution of the product from Step C (2.94 g, 6.23 mmol, 1 eq.) in THF (45 mL) and water (6 mL) was added bis(tert-butyl) dicarbonate (1.43 g, 6.55 mmol, 1.05 eq.) followed by TEA (1.73 mL, 12.47 mmol, 2 eq.) and the mixture stirred at rt for 12 h.
  • Step E 5-[2-(tert-butoxycarbonyl)-7- ⁇ [(3R)-3-methyl-3,4-dihydro-1H-isoquinolin-2- yl]carbonyl ⁇ -3,4-dihydro-1H-isoquinolin-6-yl]-1,2-dimethylpyrrole-3-carboxylic acid
  • LiOH.H2O 995 mg, 23.72 mmol, 4 eq.
  • MeOH 40 mL
  • water 20 mL
  • Step C tert-butyl 6-[4-( ⁇ 4-[(tert-butyldimethylsilyl)oxy]phenyl ⁇ (5-cyano-1,2-dimethylpyrrol-3- yl)carbamoyl)-1,5-dimethylpyrrol-2-yl]-7- ⁇ [(3R)-3-methyl-3,4-dihydro-1H-isoquinolin-2-yl]carbonyl ⁇ - 3,4-dihydro-1H-isoquinoline-2-carboxylate [634] The title compound was prepared according to General procedure 2a using Preparation IIa (2.5 g, 4.6 mmol, 1 eq.) as the appropriate acid and the product from Step B (3.14 g
  • Step D N-(5-cyano-1,2-dimethylpyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-5-(7- ⁇ [(3R)-3- methyl-3,4-dihydro-1H-isoquinolin-2-yl]carbonyl ⁇ -1,2,3,4-tetrahydroisoquinolin-6-yl)pyrrole-3- carboxamide hydrochloride [637] A solution of the product from Step C (2.8 g, 3.23 mmol, 1 eq.) in MeOH (5 mL) was treated with 3 M HCl in MeOH (10 m
  • Step B ⁇ 4-[2-(morpholin-4-yl)ethoxy]phenyl ⁇ acetic acid
  • the product from Step A (3.26 g, 11.67 mmol, 1 eq.) was hydrolysed using General procedure 8 to afford the title product (3.1 g, 11.63 mmol, quant).
  • Step A 1,2-dimethyl-5-(7- ⁇ [(3R)-3-methyl-3,4-dihydro-1H-isoquinolin-2-yl]carbonyl ⁇ -1,2,3,4- tetrahydroisoquinolin-6-yl)pyrrole-3-carboxylic acid hydrochloride
  • a solution of Preparation IIa (3 g, 5.52 mmol, 1 eq.) in MeOH (20 mL) was treated with 3 M HCl in MeOH (20 mL, 60 mmol) and stirred at rt for 3 h. The solvents were removed in vacuo and dried under vacuum to afford the title product (2.71 g, quant.).
  • Step B 1,2-dimethyl-5-(7- ⁇ [(3R)-3-methyl-3,4-dihydro-1H-isoquinolin-2-yl]carbonyl ⁇ -2-(2- ⁇ 4- [2-(morpholin-4-yl)ethoxy]phenyl ⁇ acetyl)-3,4-dihydro-1H-isoquinolin-6-yl)pyrrole-3-carboxylic acid
  • Preparation Va (1 g, 3.77 mmol, 1 eq) in anhydrous DCM (10 mL) under N2
  • 2 M oxalyl chloride solution in DCM (0.43 mL, 4.52 mmol, 1.2 eq.
  • Step A of Preparation IIa After stirring for 10 mins the product from Step A of Preparation IIa (18.6 g, 101 mmol, 1.05 eq.) was added and the mixture was stirred at rt for 1h then it was poured into water (1500 mL) and the precipitates were filtered out, washed with water.
  • This crude intermediate was dissolved in methanol (250 mL) and water (25 mL) then NaOH (28 g, 700 mmol, 7.3 eq.) was added and mixture was stirred at reflux temperature for 18 h. Evaporated at reduced pressure, then the pH was adjusted to 6 by the addition of cc. aq. HCl.
  • Step B 5-[2-(9H-fluoren-9-ylmethoxycarbonyl)-7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline- 2-carbonyl]-3,4-dihydro-1H-isoquinolin-6-yl]-1,2-dimethyl-pyrrole-3-carboxylic acid [663] To the biphasic mixture of the product from Step A (13.96 g, 28.33 mmol, 1 eq.) dissolved in dioxane (160 mL) and NaHCO3 (5.47 g, 65.2 mmol, 2.3 eq.) dissolved in water (160 mL) 9H-fluoren-9- ylmethyl carbonochloridate (8.06 g, 31.2 mmol, 1.1
  • Step D N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-N-(5-cyano-1,2-dimethyl-pyrrol-3-yl)-1,2- dimethyl-5-[7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-1,2,3,4-tetrahydroisoquinolin-6- yl]pyrrole-3-carboxamide [671] To a solution of the product from Step C (8.12 g, 8.21 mmol, 1 eq.) in DCM (41 mL) morpholine (41 mL, 475 mmol, 58 eq.) was added then the mixture was stirred at rt for 18 h.
  • Step 1 Ethyl 1,2-dimethyl-1H-pyrrole-3-carboxylate
  • ethyl 2-methyl-1H-pyrrole-3-carboxylate 10 g, 65.3 mmol
  • methyl iodide 8.95 mL, 130.6 mmol
  • sodium hydride 60 % w/w (2.6 g, 65.3 mmol)
  • the reaction mixture is stirred at 0°C for 1 hour.
  • reaction mixture is hydrolysed by addition of 420 mL of ice-cold water, diluted with ethyl acetate, and successively washed with 0.1M aqueous hydrochloric acid (HCl) solution, saturated aqueous LiCl solution and then brine.
  • HCl hydrochloric acid
  • the organic phase is dried over MgSO4, filtered, concentrated to dryness and purified by chromatography over silica gel (petroleum ether/AcOEt gradient).
  • Step 2 Ethyl 5-(5-chloro-2-formylphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylate [679] To a solution of the compound obtained in Step 1 (10.5 g, 62,8 mmol) in 65 mL of N,N- dimethylacetamide there are successively added 2-bromo-4-chlorobenzaldehyde (15.2 g, 69 mmol), potassium acetate (12.3 g, 125.6 mmol) and then the reaction mixture is stirred under argon for 20 minutes. There is added PdCl2(PPh3)2 (2.2 g, 3.14 mmol).
  • the reaction mixture is heated at 130°C overnight, allowed to cool down to room temperature and it is diluted with dichloromethane.
  • Animal charcoal is added (20g), the suspension is stirred at room temperature for 1 hour and filtered.
  • the organic phase is washed with water, dried over MgSO 4 and concentrated to dryness.
  • the crude product thereby obtained is purified by chromatography over silica gel (petroleum ether/AcOEt gradient). The title product is obtained in the form of a solid.
  • the reaction mixture is vigorously stirred at room temperature for 7 hours and concentrated to remove the acetone.
  • Ethyl acetate is added, the organic phase is washed with water, dried over MgSO 4 and then concentrated to dryness. The residue is then taken up in a minimum of ethyl ether.
  • the solid then obtained is filtered off, washed with ether and then dried in vacuo at 40°C overnight.
  • the title product is obtained in the form of a solid, which is subsequently used without being otherwise purified.
  • Step 4 ⁇ (3S)-2-[(4-Methylphenyl)sulphonyl]-1,2,3,4-tetrahydroisoquinolin-3-yl ⁇ methyl 4- methylbenzenesulphonate
  • [685] To a solution of commercially available [(3S)-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol (30.2 g, 185 mmol) in 750 mL of dichloromethane there are successively added tosyl chloride (91.7 g, ,481 mmol) and then, dropwise, N,N,N-triethylamine (122 mL, 740 mmol).
  • the reaction mixture is stirred at room temperature for 20 hours, diluted with dichloromethane, washed successively with 1M HCl solution, saturated aqueous NaHCO3 solution and then brine until neutral.
  • the organic phase is then dried over MgSO4, filtered and concentrated to dryness.
  • the solid obtained is dissolved in a minimum volume of dichloromethane and then cyclohexane is added until a precipitate is formed. This precipitate is then filtered off and washed with cyclohexane. After drying, the title product is obtained in the form of a white crystalline powder.
  • Step 5 (3S)-3-(iodomethyl)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinoline
  • sodium iodide 1. g, 9.3 mmol
  • the reaction mixture is heated under microwave irradiations (100W for 6h), cooled to room temperature.
  • the suspension is filtered.
  • the solid is washed with dichloromethane. The filtrate and the washings are pooled together and concentrated to dryness.
  • Step 6 diethyl 2-[[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3- yl]methyl]propanedioate
  • To a suspension of sodium hydride (442 mg, 11 mmol) in THF (8 mL) is added dropwise diethyl malonate (1.5 mL, 10 mmol) at room temperature. After 15 minutes, a solution of the compound obtained in Step 5 (4.2 g, 10 mmol) in THF (10 mL) is added dropwise. After 20 minutes at room temperature, the reaction mixture is added in microwave reactor (100°C-275W) during 19h.
  • the organic phase is washed with brine, dried over MgSO4 and concentrated to dryness.
  • the crude material is diluted with DMSO (10 mL) and a solution of sodium chloride (0.25 g, 4.2 mmol) in water (1 mL).
  • the reaction mixture is heated to 140°C for 1h, then cooled to room temperature and diluted with ethyl acetate.
  • the organic layer is separated, dried over MgSO 4 and concentrated to dryness, affording the desired compound.
  • Step 8 N,N-dimethyl-3-[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]propenamide
  • N-(3-Dimethylaminopropyl)-#$-ethylcarbodiimide hydrochloride 5.4 g, 28 mmol
  • 1- hydroxybenzotriazole hydrate 3.8 g, 28 mmol
  • N,N-diisopropylethylamine (6.9 mL, 41 mmol)
  • 2 M dimethylamine in THF (20.8 mL).
  • reaction mixture is stirred overnight at room temperature, diluted with dichloromethane, washed successively with water, aqueous 1M HCl solution and brine.
  • organic phase is dried over MgSO4, concentrated to dryness and purified by chromatography over silica gel using cyclohexane and ethyl acetate as eluants, affording the desired compound.
  • Step 9 N,N-dimethyl-3-[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]propan-1- amine
  • Step 8 To a solution of Step 8 (6.6 g, 17.1 mmol) in anhydrous THF (70 mL) is added dropwise under inert atmosphere a solution of 1M borane tetrahydrofuran complex in tetrahydrofuran (69 mL, 69 mmol). The reaction mixture is heated to 70°C overnight and cooled to room temperature. The reaction mixture is diluted with dichloromethane and water.
  • the organic phase is washed successively with an aqueous 1M HCl solution, a saturated aqueous NaHCO 3 solution, brine, and dried over MgSO 4 and concentrated.
  • the crude mixture is purified by chromatography over silica gel using dichloromethane and NH 3 2M in ethanol as eluants, affording the desired compound.
  • Step 10 N,N-dimethyl-3-[(3R)-1,2,3,4-tetrahydroisoquinolin-3-yl]propan-1-amine
  • naphthalene 13 g, 103 mmol
  • sodium 2.4 g, 103 mmol
  • the reaction mixture is cooled to -78°C and a solution of Step 9 (4.8 g, 12.2 mmol) in THF (35 mL) is added dropwise.
  • reaction mixture After 3 h of stirring at -78°C, the reaction mixture is allowed to warm to 0°C and a solution of saturated aqueous ammonium chloride is carefully added (3 mL). The reaction mixture is allowed to warm to room temperature and is evaporated to dryness. The crude mixture is purified by chromatography over silica gel using dichloromethane and NH 3 2M in ethanol as eluants, affording the desired compound.
  • Step 11 ethyl 5-[5-chloro-2-[(3R)-3-[3-(dimethylamino)propyl]-3,4-dihydro-1H-isoquinoline-2- carbonyl]phenyl]-1,2-dimethyl-pyrrole-3-carboxylate [707] To a solution of the compound obtained in Step 3 (15.5 g, 48 mmol) in dichloromethane (3L) are added N-(3-dimethylaminopropyl)-#$-ethylcarbodiimide hydrochloride (9.2 g, 48 mmol), 1- hydroxybenzotriazole hydrate (7.4 g, 48 mmol), the compound obtained in Step 10 (10 g, 48 mmol) and N-ethyl-N-isopropyl-propan-2-amine (28 mL, 160 mmol).
  • reaction mixture is stirred for 3h at room temperature, diluted with dichloromethane, washed with water, concentrated to dryness and purified by chromatography over silica gel using dichloromethane and NH32M in ethanol as eluants, affording the desired compound.
  • Step 12 5-[5-chloro-2-[(3R)-3-[3-(dimethylamino)propyl]-3,4-dihydro-1H-isoquinoline-2- carbonyl]phenyl]-1,2-dimethyl-pyrrole-3-carboxylic acid [711] To a solution of the compound obtained in Step 11 (400 mg, 0.76 mmol) in methanol (2 mL) is added a solution of lithium hydroxide monohydrate (112 mg, 2.6 mmol) in water (2 mL). The reaction mixture is heated at reflux overnight then partially concentrated.
  • Step 13 N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-5-[5-chloro-2-[(3R)-3-[3- (dimethylamino)propyl]-3,4-dihydro-1H-isoquinoline-2-carbonyl]phenyl]-N-(5-cyano-1,2-dimethyl- pyrrol-3-yl)-1,2-dimethyl-pyrrole-3-carboxamide [714] To a solution of the compound obtained in Step 12 (480 mg, 0.97 mmol) in 1,2-dichloroethane (60 mL) is added 1-chloro-N,N,2-trimethyl-prop-1-en-1-amine (170 ⁇ L, 1.3 mmol).
  • Step 14 5-[5-chloro-2-[(3R)-3-[3-(dimethylamino)propyl]-3,4-dihydro-1H-isoquinoline-2- carbonyl]phenyl]-N-(5-cyano-1,2-dimethyl-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-pyrrole-3- carboxamide [719] To a solution of the compound obtained in Step 13 (0.5 g, 0.61 mmol) in THF (20 mL) is added at room temperature a solution of 1M tetra-n-butylammonium fluoride in THF (1 mL, 1 mmol).
  • the reaction mixture is stirred for 4h at room temperature and diluted with dichloromethane and a saturated aqueous solution of NaHCO3.
  • the organic phase is separated, washed successively with water, brine, then dried over MgSO4 and concentrated.
  • the crude is purified by chromatography over silica gel using dichloromethane and NH32M methanol as eluants, affording the desired compound.
  • Step A N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-N-(5-cyano-1,2-dimethyl-pyrrol-3-yl)-1,2- dimethyl-5-[7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-2-[2-[4-(2- morpholinoethoxy)phenyl]acetyl]-3,4-dihydro-1H-isoquinolin-6-yl]pyrrole-3-carboxamide [725] Using the procedure described in Step D of the Preparation of P3, starting from the product of Preparation Va (1.27 g, 1.5 eq.) as the appropriate carboxylic acid and Preparation VIIb (2.45 g, 3.194 mmol) as the appropriate amine, afforded the title compound (2.06 g, 64%).
  • Step B N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-5- ⁇ 7-[(3R)- 3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl]-2-(2- ⁇ 4-[2-(morpholin-4-yl)ethoxy]phenyl ⁇ acetyl)- 1,2,3,4-tetrahydroisoquinolin-6-yl ⁇ -1H-pyrrole-3-carboxamide [728] According the procedure described in Step E of the Preparation of P3, the product from Step A (390 mg, 0.385 mmol) was treated with 2M aq.
  • NMR data were acquired at a temperature of 298K on a Bruker Avance NMR spectrometer equipped with a 5 mm BBFO CryoProbe with z-gradient operating at a frequency of 400.13 MHz for 1 H, 376.50 MHz for 19 F, 100.61 MHz for 13 C.
  • Chemical shifts for the 1 H and 13 C spectra were referenced by setting internal tetramethylsilane (TMS) to 0 ppm.
  • LC/MS data was acquired using an instrument with the following parameters: Pump Waters AcQuity UPLC Binary Solvent Manager Sample Manager Waters AcQuity UPLC Sample Manager Column Compartment Waters AcQuity UPLC Column Manager Detector Waters AcQuity UPLC PDA ELSD Shimadzu ELSD-LTII Mass Spec Waters SQD Columns AcQuity UPLC BEH C181.7 ⁇ m 2.1x50mm Eluent A1 0.1% Formic Acid in Water Eluent B1 0.1% Formic Acid in Acetonitrile Eluent A2 5mM Ammonium Hydroxide in Water Eluent B2 5mM Ammonium Hydroxide in Acetonitrile The methods used to generate LC/MS data were as follows: 2 min acidic method:
  • HRMS data was acquired using an instrument with the following parameters: HRMS_QT01 Pump Waters AcQuity UPLC Binary Solvent Manager Sample Manager Waters AcQuity UPLC Sample Manager Column Compartment Waters AcQuity UPLC Column Manager Waters AcQuity UPLC PDA n/a Waters Xevo G2 Qtof Quity UPLC PrST C4300 ⁇ 1.7 ⁇ m 2.1x100mm AcQuity UPLC CSH C181.7 ⁇ m 2.1x50mm ProSwift RP-3U 4.6x50mm SS Eluent A1 0.1% Formic Acid in Water Eluent B1 0.1% Formic Acid in Acetonitrile Eluent A2 0.05% Triflouroacetic Acid in Water Eluent B2 0.05% Triflouroacetic Acid in Acetonitrile HRMS_QT02 Pump Waters AcQuity UPLC Binary Solvent Manager Sample Manager Waters AcQuity UPLC Sample Manager Column Compartment Waters AcQuity UPLC Column Manager Waters
  • Preparative RP-HPLC Preparative-HPLC (“Prep-HPLC”) data were acquired using Teledyne ISCO purification systems using C18 or C4 RP ISCO or ISCO-gold columns.
  • Prep-HPLC methods were used: a .
  • TFA method solvent: A water + 0.05 % TFA, B acetonitrile + 0.05 % TFA, gradient from 5 to 100% B in 15 to 30 CV b .
  • NH4HCO3 method solvent: A water + 0.02 M NH4HCO3, B acetonitrile/water 80/20 + 0.02 M NH4HCO3, gradient from 5 to 100 % B in 15 to 30 CV c .
  • Neutral method solvent: A water, B acetonitrile, gradient from 5 to 100% B in 15 to 30 CV d .
  • Formic Acid method solvent: A water + 0.05 % Formic Acid, B acetonitrile + 0.05 % Formic Acid, gradient from 5 to 100% B in 15 to 30 CV Gradient variations of methods a.-d. were employed as appropriate. All the fractions containing the pure compound were combined and directly freeze-dried to afford the compound as an amorphous powder.
  • Step 1 Synthesis of 2-(bromomethyl)-4-nitrobenzoic acid
  • 2-methyl-4-nitrobenzoic acid 300 g, 1.5371 mol
  • CCl4 3000 mL
  • NBS 300.93 g, 1.6908 mol
  • AIBN 37.86 g, 0.2305 mol
  • the reaction mixture was stirred at 80°C for 16h. Reaction mixture was monitored by TLC analysis.
  • the reaction mixture was diluted with sat. NaHCO3 solution (2 lit) and extracted with ethyl acetate (2 x 2 lit). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure.
  • Step 4 Synthesis of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate, To a solution of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate (110 g, 0.4414 mol) in a mixture of EtOH (1100 mL) and H 2 O (550 mL) was added Fe Powder (197.21 g, 3.5310 mol) and NH 4 Cl (188.88 g, 3.5310 mol) at rt. The resulting mixture was heated at 80°C for 16 h. The reaction mixture was cooled to rt and filtered through celite and washed with ethylacetate (2 lit).
  • Step 5 Synthesis of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol
  • THF 1000 mL
  • LiAlH 4 (1 M in THF)
  • 798.2 mmol 798.2 mL
  • a solution of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate 70 g, 319.3 mmol
  • the reaction was stirred at rt for 4 h.
  • the reaction mixture was cooled to 0°C, then was added water (22 mL) very slowly and followed by the addition of 20% NaOH (22 mL) and water (66 mL). The reaction mixture was stirred at 0°C for 30 min. Anhydrous sodium sulphate was added to absorb excess of water. The mixture was filtered through celite. The filter cake was washed with ethylacetate (1000 mL) and 10% MeOH/DCM (500 mL). The filtrate was concentrated under reduced pressure.
  • Step 6 Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1- yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate
  • (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (1.92 g, 10.04 mmol, 1.0 equiv.)
  • (9H-fluoren-9-yl)methyl (S)-(1-amino-1-oxo-5-ureidopentan-2-yl)carbamate (3.99 g, 10.04 mmol, 1.0 equiv.)
  • (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (4.20 g, 11.04 mmol,
  • Step 7 Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5- ureidopentanamide
  • (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1- yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (608 g, 10.65 mmol, 1.0 equiv.) was added dimethylamine (2 M in THF, 21.31 mL, 42.62 mmol, 4 equiv.).
  • Step 8 Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1- yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
  • (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5- ureidopentanamide (3.50 g, 10.04 mmol, 1.0 equiv.)
  • Step 1 Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid
  • MeOH 1000 mL
  • KOH 28.19 g, 502.43 mmol, 1.00 eq
  • H2O 150 mL
  • Step 2 Synthesis of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol
  • 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid 41 g, 94.14 mmol, 1 eq
  • THF 1 M, 470.68 mL, 5 eq
  • the yellow mixture was stirred at 60°C for 2h.
  • Step 3 Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde
  • Step 4 Synthesis of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine
  • 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (12.6 g, 30.03 mmol, 1 eq) in DCM (130 mL) was added prop-2-yn-1-amine (4.14 g, 75.08 mmol, 4.81 mL, 2.5 eq) and MgSO4 (36.15 g, 300.
  • Step 5 Synthesis of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5- nitrobenzyl)(prop-2-yn-1-yl)carbamate
  • a solution of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 19.62 mmol, 1 eq) and Fmoc-OSU (7.28 g, 21.59 mmol, 1.1 eq) in dioxane (90 mL) was added sat. NaHCO3 (90 mL) and the white suspension was stirred at 20°C for 12hr.
  • Step 6 Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate
  • 9H-fluoren-9-yl)methyl (2-(((tert- butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate 5.0 g, 7.34 mmol, 1.0 equiv.
  • 10% AcOH/CH2Cl2 100 mL
  • Step 7 Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1- yl)carbamate
  • (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn- 1-yl)carbamate (2.99 g, 4.59 mmol, 1.0 equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5
  • Step 8 Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate
  • To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate
  • 9H-fluoren-9-yl)methyl 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)
  • Step 9 Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate
  • To a solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1- yl)carbamate (984 mg, 1.135 mmol, 1.0 equiv.) in THF (7.5 mL) was added 1.0 M tetrabutylammoniumn fluoride in THF (2.27 mL,
  • Step 1 Synthesis of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide To a stirred suspension of 6-nitroisobenzofuran-1(3H)-one (500 g, 2.79 mol) in MeOH (1500 mL) was added MeNH2 (3.00 kg, 29.94 mol, 600 mL, 31.0% purity) at 25 °C and stirred for 1 h.
  • Step 2 Synthesis of (2-((methylamino)methyl)-4-nitrophenyl)methanol
  • a solution of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, 2.66 mol) in THF (5000 mL) was cooled to 0 °C, then BH 3 -Me 2 S (506 g, 6.66 mol) (2.0 M in THF) was added drop wise for 60 min and the mixture was heated to 70 °C for 5 h.
  • LCMS showed the starting material was consumed.
  • 4M HCl (1200 mL) in Methanol was added to the reaction mixture at 0 °C and heated at 65 °C for 8 h.
  • Step 4 Synthesis of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5- nitrobenzyl)(methyl)carbamate
  • Fmoc-OSU 341.5 g, 1.01 mol
  • Et 3 N 186.2 g, 1.84 mol, 256.2 mL
  • Step 5 Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate
  • Step 6 Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate
  • (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.86 g, 4.56 mmol, 1.0 equiv)
  • (S)-2-((S)- 2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (1.71 g, 4.56 mmol, 1.0 equiv.) in 2:1 CH2Cl2/MeOH (60 m
  • Step 7 Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate
  • To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.05 g, 2.085 mmol, 1.0 equiv) in THF (10 mL) was added 2.0 M dimethyl amine in MeOH (10.42 mL, 20.85 mmol, 10 equiv.).
  • Step 2 Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide
  • Step 3 Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5- ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
  • (S)-2-amino-N-(4-(hydroxymethyl)-3-nitrophenyl)-5-ureidopentanamide (10.89 g, 28.8 mmol, 1.0 equiv.)
  • 1-hydroxy- 7-azabenzotriazole (3.92 g, 28.8 mmol, 1.0 equiv.) in DMF (40 mL) was added 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide (5.52 g
  • Step 4 Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3- nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate
  • a suspension of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-nitrophenyl)amino)-1-oxo-5- ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 11.96 g, 22.8 mmol, 1.0 equiv.
  • imidazole 15.52 g, 228 mmol, 10 equiv.
  • Step 5 Synthesis of tert-butyl ((S)-1-(((S)-1-((3-amino-4-(((tert- butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan- 2-yl)carbamate
  • tert-butyl ((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)-3- nitrophenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8.02 g, 12.56 mmol, 1.0 equiv.) in methanol (250 mL) under a nitrogen atmosphere was added palladium on carbon (10 wt%, 2.00
  • Step 6 Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-(2-(((prop-2-yn-1- yloxy)carbonyl)amino)acetamido)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1- oxobutan-2-yl)carbamate

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Abstract

L'invention concerne des conjugués anticorps-médicament qui se lient à des cibles oncologiques humaines. Les conjugués anticorps-médicament comprennent un anticorps ou un fragment de liaison à l'antigène de celui-ci lié de manière covalente à au moins un mimétique BH3 par l'intermédiaire d'un lieur double et le fragment de liaison à l'antigène de celui-ci se lie à l'antigène cible CD7. L'invention concerne en outre des méthodes et des compositions destinées à être utilisées dans le traitement de cancers par administration des conjugués anticorps-médicament décrits ici. L'invention concerne également des conjugués lieur-médicament comprenant au moins un mimétique BH3 et leurs méthodes de fabrication.
PCT/US2024/056834 2023-11-22 2024-11-21 Conjugués anticorps-médicament anti-cd7 et leurs procédés d'utilisation Pending WO2025111431A1 (fr)

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