Anti-CD74 antibody-drug conjugates and methods of use thereof

Abstract

Anti-CD74 antibody-drug conjugates are disclosed. The antibody-drug conjugates comprise an anti-CD74 antibody or an antigen-binding fragment thereof covalently linked to two antineoplastic payloads through a dual linker. The disclosure further relates to methods and compositions for use in the treatment of cancers by administering the antibody-drug conjugates provided herein.

Description

[0001] 132043-01120 ANTI-CD74 ANTIBODY-DRUG CONJUGATES AND METHODS OF USE THEREOF RELATED APPLICATION

[0001] This application claims the benefit of the filing date, under 35 U.S.C. §119(e), of U.S.Provisional Application No. 63 / 601,934, filed on November 22, 2023 and European Patent Application No. 23211616.0, filed on November 22, 2023, the entire contents of which are incorporated here by reference. SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XMLformat and is hereby incorporated by reference in its entirety. Said .XML copy, created on November 19, 2024, is named “132043-01120.xml” and is 33,747 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. FIELD OF THE INVENTION

[0003] The present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-CD74antibody or an antigen-binding fragment thereof covalently linked to two specific antineoplastic compounds through a dual linker. The disclosure further relates to methods and compositions useful in the treatment and / or diagnosis of cancers that express a target antigen 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. BACKGROUND OF THE INVENTION

[0004] Apoptosis (programmed cell death) is an evolutionarily conserved pathway essential for tissuehomeostasis, 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, Annu Rev Immunol.2003;21:71-105; Vaux and Flavell, Curr Opin Immunol. 2000 Dec;12(6):719-24). Evasion of apoptosis is recognized as a hallmark of cancer, participating in the development as well as the sustained expansion of tumors and the resistance to anti-cancer treatments (Hanahan and Weinberg, Cell.2000 Jan 7;100(1):57-70).

[0005] 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(1):47-59). 1 ME150999036v.1 132043-01120

[0006] In the face of stress stimuli, whether a cell survives or undergoes apoptosis is dependent onthe extent of pairing between the Bcl-2 family members that promote cell death with family members that promote cell survival. For the most part, these interactions involve the docking of the Bcl-2 homology 3 (BH3) domain of proapoptotic family members into a groove on the surface of pro- survival members. The presence of Bcl-2 homology (BH) domain defines the membership of the Bcl- 2 family, which is divided into three main groups depending upon the particular BH domains present within the protein. 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(1):47-59).

[0007] Overexpression of the prosurvival members of the Bcl-2 family is a hallmark of cancer and ithas been shown that these proteins play an important role in tumor development, maintenance andresistance to anticancer therapy (Czabotar et al., Nat. Rev. Mol. Cell Biol. 2014 Jan;15(1):49-63). Bcl-xL (also named BCL2L1, from BCL2-like 1) is frequently amplified in cancer (Beroukhim et al., Nature 2010 Feb 18;463(7283):899-905) and it has been shown that its expression inversely correlates with sensitivity to more than 120 anti-cancer therapeutic molecules in a representative panel of cancer cell lines (NCI-60) (Amundson et al., Cancer Res.2000 Nov 1;60(21):6101-10).

[0008] In addition, several studies using transgenic knockout mouse models and transgenicoverexpression of Bcl-2 family members highlighted the importance of these proteins in the diseases of the immune system and autoimmune diseases (for a review, see Merino et al., Apoptosis 2009 Apr;14(4):570-83. doi: 10.1007 / s10495-008-0308-4.PMID: 19172396). Transgenic overexpression of Bcl-xL within the T-cell compartment resulted in resistance to apoptosis induced by glucocorticoid, g- radiation and CD3 crosslinking, suggesting that transgenic Bcl-xL overexpression can reduce apoptosis in resting and activated T-cells (Droin et al., Biochim Biophys Acta 2004 Mar 1;1644(2- 3):179-88. doi: 10.1016 / j.bbamcr.2003.10.011.PMID: 14996502 ). In patient samples, persistent or high expression of antiapoptotic Bcl-2 family proteins has been observed (Pope et al., Nat Rev Immunol. 2002 Jul;2(7):527-35. doi: 10.1038 / nri846.PMID: 12094227). In particular, 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).

[0009] The findings indicated above motivated the discovery and development of a new class of drugsnamed 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. 2012 Feb 10;30(5):488-96). After that, selective inhibitors of Bcl-2 (ABT-199 and S55746 – Souers et al., Nat Med. 2013 Feb;19(2):202-8; Casara et 2 ME150999036v.1 132043-01120 al., Oncotarget 2018 Apr 13;9(28):20075-20088), Bcl-xL (A-1155463 and A-1331852 - Tao et al., ACS Med Chem Lett.2014 Aug 26;5(10):1088-93; Leverson et al., Sci Transl Med.2015 Mar 18;7(279):279ra40) and Mcl-1 (A-1210477, S63845, S64315, AMG-176 and AZD-5991 - Leverson et al., Cell Death Dis. 2015 Jan 15;6:e1590.; Kotschy et al., Nature 2016, 538, 477-482; Maragno et al., AACR 2019, Poster #4482; Kotschy et al., WO 2015 / 097123; Caenepeel et al., Cancer Discov. 2018 Dec;8(12):1582-1597; Tron et al., Nat. Commun.2018 Dec 17;9(1):5341) were also discovered. 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. In pre-clinical models, ABT-263 has shown activity in several hematological malignancies and solid tumors (Shoemaker et al., 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). In clinical studies, 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-1155463 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.2014 Aug 26;5(10):1088-93; Leverson et al., Sci. Transl. Med.2015 Mar 18;7(279):279ra40). The use of BH3 mimetics has also shown benefit in pre- clinical models of diseases of the immune system and autoimmune diseases. Treatment with ABT-737 (Bcl-2, Bcl-xL, and Bcl-w inhibitor) resulted in potent inhibition of lymphocyte proliferation in vitro. Importantly, mice treated with ABT-737 in animal models of arthritis and lupus showed a significant decrease in disease severity (Bardwell et al., J Clin Invest. 1997 Feb 1;99(3):439-46. doi: 10.1172 / JCI119178.PMID: 9022077). In addition, it has been shown that 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 2011 May 25.PMID: 21615547).

[0010] In pre-clinical studies, it has been shown that BH3 mimetics strongly synergize when incombination, including Mcl1i + Bcl2i, Mcl1i + Bcl-xli, Bcl-xli + Bcl-2i (WO 2018015526A1; Moujalled et al., Leukemia. 2019 Apr;33(4):905-917; Moujalled et al., Blood Adv. 2020 Jun 23;4(12):2762-2767; Grundy et al., Oncotarget. 2018 Dec 28;9(102):37777-37789; Soderquist et al., Nat Commun. 2018 Aug 29;9(1):3513; Weeden et al., Oncogene. 2018 Aug;37(32):4475-4488; Sarah Kehr et al., Cancer Lett. 2020 Jul 10;482:19-32). Furthermore, it has also been shown that Bcl-xl inhibitors and Mcl1 inhibitors strongly synergize when in combination with taxane (Leverson et al, Science Translation Medicine, 2015 March 18 ; Vol 7(279) 279ra40 ; Bah et al, Cell Death and Disease, 20145, e1291; Wong et al, Mol Cancer Ther., 2012 Apr; 11(4) 1026-1035; Bennett et al, Open Biol., 20166: 160134; Topham et al, Cancer Cell, 201528, 129-140; Nguyen et al, Clin Cancer Res, 2011 3 ME150999036v.1 132043-01120 March 15, 17(6) 1394-1404; Merino et al, Science Translational Medicine, 2017 Aug2;9(401):eaam7049). Even if the activity of these combinations is very promising, evidence of toleranceof the administration of two non-conjugated BH3 mimetics in combination is still missing. Also, the clinical potential of non-conjugated BH3 mimetics combinations is still to be demonstrated.

[0011] CD74 (DHLAG) is an established and attractive target for antibody drug conjugates due to itsrestricted expression on normal tissues and significant upregulation in a range of hematological malignancies. CD74 functions as a chaperone that is necessary for the assembly and trafficking of MHC class II complexes as well as a receptor for macrophage migration inhibitory receptor (MIF). In oncology, it has been well established that CD74 is significantly upregulated at both the RNA and protein level in a range of B-cell and myeloid cell malignancies including acute myeloid leukemia (AML), multiple myeloma, and diffuse large B-cell lymphoma. Additionally CD74 is known to rapidly internalize upon antibody engagement and traffic to the lysosome as well as to be rapidly repopulated on the surface of tumor cells following internalization. Antibodies and antibody drug conjugates targeting CD74 have been shown previously to demonstrate anti-tumor activity in preclinical models of cancer.

[0012] Therefore, there is need to find disease-modifying agents therapeutically targeting CD74, Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL, Mcl-1) or upstream and / or downstream proteins in an apoptotic signaling pathway in oncology and in the field of immune and autoimmune diseases. SUMMARY OF THE INVENTION

[0013] In one embodiment, the present disclosure provides an antibody-drug conjugate of Formula(I), (I), wherein is selected from the group consisting of: 4 ME150999036v.1 132043-01120 5 ME150999036v.1 132043-01120 wherein indicates the point of attachment to Ab; wherein Ab is an anti-CD74 antibody or an antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) and three light chain CDRs selected from the group consisting of: 1) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:12; 2) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:4, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; 6 ME150999036v.1 132043-01120 3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:6, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:7, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:8; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; and 4) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:9, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; and wherein a is an integer from 1 to 16.

[0014] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18. The definitions of the remaining variables are provided in any other embodiments described herein.

[0015] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof comprises: 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:20 or a sequence that is at least 95% identical to SEQ ID NO:20. The definitions of the remaining variables are provided in any other embodiments described herein.

[0016] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof comprises one or more Fc silencing mutations. The definitions of the remaining variables are provided in any other embodiments described herein.

[0017] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof comprise one or more CysMab mutations. The definitions of the remaining variables are provided in any other embodiments described herein.

[0018] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof comprises one or more CysMab mutations selected from E152C, S375C, or both E152C and S375C of the heavy chain of the antibody or antigen binding fragment thereof, wherein the position is numbered according to the EU system. The definitions of the remaining variables are provided in any other embodiments described herein.

[0019] In another embodiment, the present disclosure provides an antibody-drug conjugate, wherein7 ME150999036v.1 132043-01120 the anti-CD74 antibody or antigen-binding fragment thereof comprises a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a single chain Fv (scFv), a disulfide-linked Fv (sdFv), a Fd fragment, a Fv fragment, a dAb fragment, a maxibody, a minibody, an intrabody, a diabody, a half antibody, or a one-arm antibody. The definitions of the remaining variables are provided in any other embodiments described herein.

[0020] In another embodiment, the present disclosure provides an antibody-drug conjugate, whereinthe anti-CD74 antibody or antigen-binding fragment thereof is a monoclonal antibody. The definitions of the remaining variables are provided in any other embodiments described herein.

[0021] In another embodiment, the present disclosure provides an antibody-drug conjugate, wherein The definitions of the remaining variables are provided in any other embodiments described herein.

[0022] In another embodiment, the present disclosure provides an antibody-drug conjugate of,wherein is: The definitions of the remaining variables are provided in any other embodiments described herein.

[0023] In another embodiment, the present disclosure provides an antibody-drug conjugate, wherein8 ME150999036v.1 132043-01120 . The definitions of the remaining variables are provided in any other embodiments described herein.

[0024] In another embodiment, the present disclosure provides an antibody-drug conjugate of,wherein is: The definitions of the remaining variables are provided in any other embodiments described herein.

[0025] In another embodiment, the present disclosure provides an antibody-drug conjugate, wherein 9 ME150999036v.1 132043-01120 . The definitions of the remaining variables are provided in any other embodiments described herein.

[0026] In another embodiment, the present disclosure provides an antibody-drug conjugate, wherein . The definitions of the remaining variables are provided in a any other embodiment described herein.

[0027] In another embodiment, the present disclosure provides an antibody-drug conjugate of anantibody-drug conjugate, wherein is: . The definitions of the remaining variables are provided in any other embodiment described herein.

[0028] In some embodiments, a is an integer from 1 to 8. In some embodiments, a is an integer from1 to 5. In some embodiments, a is an integer from 2 to 4. In some embodiments, a is 2. In some 10 ME150999036v.1 132043-01120 embodiments, a is 4. In some embodiments, a is determined by liquid chromatography-mass spectrometry (LC-MS).

[0029] In some embodiments, the anti-CD74 antibody is VHmil x VK1aNQ.

[0030] In some embodiments, the antibody or antigen-binding fragment binds to a target antigen on acancer cell. In some embodiments, the target antigen is CD74.

[0031] In some embodiments, the present disclosure provides an antibody or antigen-bindingfragment comprising one or more SEQ IDs listed in Tables A2 and A3 described herein.

[0032] Also provided herein, in some embodiments, are compositions comprising multiple copies ofan antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein). In some embodiments, the average a of the antibody-drug conjugates in the composition is from about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 2, or about 2 to about 4.

[0033] Also provided herein, in some embodiments, are pharmaceutical compositions comprising anantibody-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.

[0034] Further provided herein, in some embodiments, are therapeutic uses for the describedantibody-drug conjugate compounds and compositions, e.g., in treating a cancer. In some embodiments, 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 antibody-drug conjugate, such as CD74). In some embodiments, 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 antibody-drug conjugate compound or composition disclosed herein.

[0035] An exemplary embodiment is a method of treating a subject having or suspected of having acancer, 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). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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, 11 ME150999036v.1 132043-01120 pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0036] Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumorin 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). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, 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. In some embodiments, 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%.

[0037] Another exemplary embodiment is a method of reducing or inhibiting a hematological cancerin 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). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, 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). In some embodiments, administration of the antibody-drug conjugate, 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%.

[0038] Another exemplary embodiment is a method of reducing or slowing the expansion of a cancercell 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). In some embodiments, the cancer cell population expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer cell population is from a tumor or a hematological cancer. In some embodiments, the cancer cell population is from a breast 12 ME150999036v.1 132043-01120 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. In some embodiments, the cancer cell population is from acute myeloid leukemia, multiple myeloma, or B-cell lymphoma. In some embodiments, 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%. In some embodiments, 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%.

[0039] Another exemplary embodiment is an antibody-drug conjugate, composition, orpharmaceutical 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. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0040] Another exemplary embodiment is a use of an antibody-drug conjugate, composition, orpharmaceutical 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. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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, 13 ME150999036v.1 132043-01120 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0041] Another exemplary embodiment is a use of an antibody-drug conjugate, composition, orpharmaceutical 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. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0042] Another exemplary embodiment is a method of determining whether a subject having orsuspected 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. In some embodiments, the cancer cells in the sample express a target antigen. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. 14 ME150999036v.1 132043-01120

[0043] Methods of producing the described antibody-drug conjugate compounds and compositions(e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein) are also disclosed. An exemplary embodiment is a method of producing an antibody-drug conjugate by conjugating an anti-CD74 antibody or antigen-binding fragment to a dual linker joined or covalently attached to two antineoplastic compounds described herein under conditions that allow conjugation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 depicts a complex structure of VHmil x Vk1aNQ Fab and CD74 peptide showing theFab as ribbon representation and the bound peptide as sticks with its molecular surface (Connolly surface). The heavy chain is shown in dark grey, the light chain is shown in white and the peptide is colored in light grey. The terminal amino acids of the peptide chain are labelled.

[0045] FIG. 2 is an unbiased Fo-Fc omit map contoured at 4.4σ which is shown with the final refinedCD74 peptide model as ball-and-stick representation superimposed. Only for the eight C-terminal residues (AA289 to AA296) strong and clear electron density was revealed. No interpretable electron density was observed for the remaining eight N-terminal residues (AA281 to AA288).

[0046] FIG. 3 is a graph showing the number of direct intermolecular contacts (# of non-H mABatoms within 3.8 Å and the reduction in solvent accessible surface (Å2) upon binding plotted per CD74-peptide amino acid (X-axis).

[0047] FIG. 4A is a graph showing the number of direct intermolecular contacts (# of non-H mABatoms within 3.8 Å and the reduction in solvent accessible surface (Å2)) upon binding plotted per amino acid from the VHmil x Vk1aNQ Fab heavy chain (SEQ ID NO:24) (X-axis).

[0048] FIG. 4B is a graph showing the number of direct intermolecular contacts (# of non-H mABatoms within 3.8 Å and the reduction in solvent accessible surface (Å2)) upon binding plotted per amino acid from the VHmil x Vk1aNQ Fab light chain (SEQ ID NO: 25) (X-axis)).

[0049] FIG. 5 is a graph showing growth inhibition (GI50) of the CD74 (VHmil x Vk1aNQ) -P1-L12-P4 ADC in µM for the representative cell lines tested.

[0050] FIG. 6 are graphs showing dose response curves of eight ADCs - VHmil x VK1aNQ-P1-L12-P3, VHmil x VK1aNQ-P1-L17-P2, VHmil x VK1aNQ-P1-L12-P2, VHmil x VK1aNQ-P1-L17-P3, VHmil x VK1aNQ-P1-L17-P4, VHmil x VK1aNQ-P1-L19-P2, VHmil x VK1aNQ-P1-L12-P4, IgG- P1-L12-P4 in the Nomo1, EOL1, and Monomac1 cell lines.

[0051] Fig. 7A-7D are graphs showing efficacy data generated in a disseminated EOL1-Luc tumormodel treated with a CD74-MCL-1 / BCL-2 dual ADC (VHmil x VK1aNQ-P1-L12-P4 ADC). Mice were also treated with an isotype control ADC (IgG-P1-L12-P4 ADC). ADCs were administered on Q2Wx2 schedule. EOL-1 tumors Leukemic burden was evaluated using a Xenogen bioluminescence imager. Group averages are presented in (7A), and individual tumor data is presented in (7B-7D). 15 ME150999036v.1 132043-01120 DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0052] The disclosed compositions and methods may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanying figures, which form a part of this disclosure.

[0053] Throughout this text, the descriptions refer to compositions and methods of using thecompositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using the composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

[0054] When a range of values is expressed, it includes embodiments using any particular valuewithin the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and / or” unless the specific context of its use dictates otherwise. All references cited herein are incorporated by reference for any purpose. Where a reference and the specification conflict, the specification will control.

[0055] Unless the context of a description indicates otherwise, e.g., in the absence of symbolsindicating specific point(s) of connectivity, when a structure or fragment of a structure is drawn, it may be used on its own or attached to other components of an antibody-drug conjugate (ADC), and it may do so with any orientation, e.g., with the antibody attached at any suitable attachment point to a chemical moiety such as a linker-drug. Where indicated, however, components of an ADC are attached in the orientation shown in a given formula.

[0056] It is to be appreciated that certain features of the disclosed compositions and methods, whichare, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0057] As used throughout this application, antibody drug conjugates can be identified using anaming convention in the general format of “target antigen / antibody-payload-dual linker-payload.” The term “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., a BH3 mimetic) described here unless otherwise specified and L# refers to a specific dual linker unless otherwise specified. For example only, if an antibody drug conjugate is referred to as “Target X-P1-L1-P2”, such a conjugate would 16 ME150999036v.1 132043-01120 comprise an antibody that binds Target X, a dual linker designated as L1, and two payloads designated as P1 and P2, respectively, including an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and / or pharmaceutically acceptable salt of any of the foregoing. Alternatively, if 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. In another alternative, if 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”. Exemplary embodiments of the present disclosure include VHmil x VK1aNQ-P1-L12-P2, VHmil x VK1aNQ-P1-L12-P3, VHmil x VK1aNQ-P1-L12- P4, VHmil x VK1aNQ-P1-L17-P2, VHmil x VK1aNQ-P1-L17-P3, VHmil x VK1aNQ-P1-L17-P4, and VHmil x VK1aNQ-P1-L19-P2.

[0058] Any formula given herein is also intended to represent unlabeled forms as well as isotopicallylabeled 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 as3H,11C,13C,14C,15N,18F, and36Cl. 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 as3H and14C, or those into which non-radioactive isotopes, such as2H and13C are present. Such isotopically labelled compounds are useful in metabolic studies (with14C), reaction kinetic studies (with, for example2H or3H), 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. In particular, an18F 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. Definitions

[0059] Various terms relating to aspects of the description are used throughout the specification andclaims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the contextclearly dictates otherwise. The terms “comprising”, “having”, “being of” as in “being of a chemical formula”, “including”, and “containing” are to be construed as open terms (i.e., meaning “including 17 ME150999036v.1 132043-01120 but not limited to”) unless otherwise noted. Additionally whenever “comprising” or another open- ended term is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of” or the closed term “consisting of”.

[0061] The term "about" or "approximately," when used in the context of numerical values andranges, refers to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, as is apparent to the skilled person from the teachings contained herein. In some embodiments, about means plus or minus 20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% of a numerical amount. 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.

[0062] The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,”“immunoconjugate,” and “ADC” are used interchangeably, and refer to one or more therapeutic compounds described herein that is linked to one or more anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the ADC is defined by the generic formula: (Formula I), wherein Ab = an anti-CD74 antibody or antigen-binding fragment, L = a dual linker moiety, D1and D2= a drug moiety (e.g., a Mcl-1 inhibitor , Bcl-2 inhibitor, Bcl-xL inhibitor drug moiety), and a = the number of dual linker moieties with attached D1and D2per antibody or antigen- binding fragment. In ADCs comprising antineoplastic payloads described herein, “2a” refers to the number of antineoplastic payloads described herein linked to the antibody or antigen-binding fragment.

[0063] The term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule thatrecognizes 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 18 ME150999036v.1 132043-01120 regions (FR). 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.

[0064] In some embodiments, the antibody or antibody fragment disclosed herein include modifiedor 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). In one embodiment, 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. Methods for inserting cysteine at specific locations of an antibody are known in the art, see, e.g., Lyons et al., (1990) Protein Eng., 3:703-708, WO 2011 / 005481, WO2014 / 124316, WO 2015 / 138615. In certain embodiments, 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. In some embodiments 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. In certain embodiments 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. In certain embodiments 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 19 ME150999036v.1 132043-01120 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. In particular embodiments 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. In particular embodiments 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. In other particular embodiments 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.

[0065] The term “antibody fragment” or “antigen-binding fragment” or “functional antibodyfragment,” 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., CD74). 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. It has been shown that fragments of a full-length antibody can perform the antigen binding function of a full-length antibody. Examples of 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). The term “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 20 ME150999036v.1 132043-01120 the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, 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.

[0066] The term “complementarity determining region” or “CDR,” as used herein, refers to thesequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). 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. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al. (1997) J Mol Biol. 273(4):927-48 (“Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc (2001) Nucleic Acids Res. 29(1):207-9; Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77) (“IMGT” numbering scheme); or a combination thereof. In a combined Kabat and Chothia numbering scheme, 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. As used herein, the CDRs defined according to the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”

[0067] In some embodiments, under Kabat, the CDR amino acid residues in the heavy chain variabledomain (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). In some embodiments, under Chothia, 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). By combining the CDR definitions of both Kabat and Chothia, in some embodiments, 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. In some embodiments, under IMGT, 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). In some embodiments, under IMGT, the CDR regions of an antibody may be determined using the program IMGT / DomainGap Align. 21 ME150999036v.1 132043-01120

[0068] The term "monoclonal antibody," as used herein, refers to an antibody obtained from apopulation 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. For example, 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.

[0069] The monoclonal antibodies described herein can be non-human, human, or humanized. Theterm 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.

[0070] The term “human antibody,” as used herein, refers an antibody produced by a human or anantibody 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). The structures and locations of immunoglobulin variable domains, e.g., 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). However, the term “human antibody,” as used herein, is not intended to include 22 ME150999036v.1 132043-01120 antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0071] The term “recombinant human antibody,” as used herein, refers to a human antibody that isprepared, 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. In some embodiments, however, 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.

[0072] The term “chimeric antibody,” as used herein, refers to antibodies wherein the amino acidsequence of the immunoglobulin molecule is derived from two or more species. In some instances, 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.

[0073] As used herein, the term "humanized antibody" refers to forms of antibodies that containsequences 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. In general, 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. 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.

[0074] “Internalizing” as used herein in reference to an antibody or antigen-binding fragment refersto 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. For example, an internalizing anti-CD74 antibody is one 23 ME150999036v.1 132043-01120 that is capable of being taken into the cell after binding to CD74 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., CD74) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC transfers through the cellular membrane after antigen binding). In some embodiments, 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. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is taken into the cell via receptor- mediated endocytosis.

[0075] “Non-internalizing” as used herein in reference to an antibody or antigen-binding fragmentrefers to an antibody or antigen-binding fragment that remains at the cell surface upon binding to the cell. In some embodiments, 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).

[0076] The term “cluster of differentiation 74” or “CD74,” as used herein, refers to any native formof human CD74 (also known as HLA class II histocompatibility antigen gamma chain or HLA-DR antigens-associated invariant chain). The term encompasses full-length human CD74 (e.g., NCBI Reference Sequence: NP_001020330.1; SEQ ID NO:22), as well as any form of human CD74 that may result from cellular processing. The term also encompasses functional variants or fragments of human CD74, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CD74 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). CD74 can be isolated from human, or may be produced recombinantly or by synthetic methods.

[0077] The term “anti-CD74 antibody” or “antibody that binds to CD74,” as used herein, refers toany form of antibody or antigen-binding fragment thereof that binds, e.g., specifically binds, to CD74. 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 CD74. WO2020 / 236817 provides and is incorporated herein by reference for exemplary CD74-binding sequences, including exemplary anti-CD74 antibody sequences. In some embodiments, the anti-CD74 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antigen-binding fragment. VHmil x VK1aNQ (WO2020 / 236817) is an exemplary anti-CD74 antibody.

[0078] The term “binding specificity,” as used herein, refers to the ability of an individual antibodyor 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. Also, as used 24 ME150999036v.1 132043-01120 herein, the terms "specific," "specifically binds," and "binds specifically" refer to a binding reaction between an antibody or antigen-binding fragment (e.g., an anti-CD74 antibody) and a target antigen (e.g., CD74) 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., CD74), but does not bind (or exhibits minimal binding) to other antigens. In some embodiments, an antibody or antigen-binding fragment that specifically binds a target antigen (e.g., CD74) has a KD of less than 1x10-6M, less than 1x10-7M, less than 1x10-8M, less than 1x10-9M, less than 1x10-10M, less than 1x10-11M, less than 1x10-12M, or less than 1x10-13M. In some embodiments, the KDis 1 pM to 500 pM. In some embodiments, the KDis between 500 pM to 1 µM, 1 µM to 100 nM, or 100 mM to 10 nM.

[0079] The term “affinity,” as used herein, refers to the strength of interaction between antibody andantigen 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.

[0080] The term "kon" or "ka" refers to the on-rate constant for association of an antibody to theantigen 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.

[0081] The term "koff" or "kd" refers to the off-rate constant for dissociation of an antibody from theantibody / antigen complex. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.

[0082] The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigeninteraction. KDis calculated by ka / kd. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.

[0083] The term “epitope” refers to the portion of an antigen capable of being recognized andspecifically 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 (or antigen-binding fragment) may be identified using any epitope 25 ME150999036v.1 132043-01120 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).

[0084] Competitive binding and epitope binning can also be used to determine antibodies sharingidentical 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 (1stedition 1988, 2ndedition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as CD74 (e.g., a binding protein comprising CDRs and / or variable domains selected from those identified in Tables A2 and A3), 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. In some embodiments, 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)). In some embodiments, 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.

[0085] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably torefer 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. The terms also include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0086] A "recombinant” protein refers to a protein (e.g., an antibody) made using recombinanttechniques, e.g., through the expression of a recombinant nucleic acid. 26 ME150999036v.1 132043-01120

[0087] An "isolated" protein refers to a protein unaccompanied by at least some of the material withwhich it is normally associated in its natural state. For example, a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the living organism, is isolated. The definition includes the production of an antibody in a wide variety of organisms and / or host cells that are known in the art.

[0088] An "isolated antibody," as used herein, is an antibody that has been identified and separatedfrom 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. In some embodiments, 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 are known in the art and include, without limitation, protein A chromatography, anion exchange chromatography, cation exchange chromatography, virus retentive filtration, and ultrafiltration.

[0089] As used herein, the term “variant” refers to a nucleic acid sequence or an amino acid sequencethat 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. In some embodiments, 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. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the unmodified peptide and the variant are closely similar overall and, in many regions, identical. In some embodiments, a peptide variant retains one or more functional properties of the unmodified peptide sequence. A variant and unmodified peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.

[0090] A variant of a nucleic acid or peptide can be a naturally-occurring variant or a variant that isnot 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. As long as a sequence contains a different nucleic acid or amino acid as compared to a reference sequence, it is considered a “variant” regardless of how it was synthesized. In some embodiments, a variant has high sequence identity (i.e., 60% nucleic acid or amino acid sequence identity or higher) as compared to a reference sequence. In some embodiments, a peptide variant encompasses polypeptides having amino acid 27 ME150999036v.1 132043-01120 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. In some embodiments, 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.

[0091] The term “conservatively modified variant” applies to both amino acid and nucleic acidsequences. For 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. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) 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. For polypeptide sequences, 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.

[0092] The term “conservative sequence modifications,” as used herein, refers to amino acidmodifications 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. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains 28 ME150999036v.1 132043-01120 (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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, 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.

[0093] The term “homologous” or “identity,” as used herein, refers to the subunit sequence identitybetween 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.

[0094] Percentage of “sequence identity” can be determined by comparing two optimally alignedsequences 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. Generally, the amino acid identity or homology between proteins disclosed herein and variants thereof, including variants of target antigens (such as CD74) 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%.

[0095] The comparison of sequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. In some embodiments, 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 29 ME150999036v.1 132043-01120 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. In some embodiments, 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.

[0096] The term “agent” is used herein to refer to a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, an extract made from biological materials, or a combination of two or more thereof. The term “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.

[0097] The term "chemotherapeutic agent" or “anti-cancer agent” is used herein to refer to all agentsthat 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 mimetics and ADCs comprising them, as described herein. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell growth and / or multiplication of cells. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with a cell’s expression activity and / or functioning.

[0098] The term “antineoplastic payload” or “antineoplastic compound” as used herein, refers to acompound or compounds that slow or inhibit the division of cancerous cells or that kill the cancerous cells. Non-limiting examples of antineoplastic payloads include the drugs described herein.

[0099] The term “BH3 mimetic,” as used herein refers to an agent capable of disrupting theinteraction 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.

[0100] The term “myeloid cell leukemia 1” or “Mcl-1,” as used herein, refers to any native form ofhuman Mcl-1, an anti-apoptotic member of the Bcl-2 protein family. 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. The term also encompasses functional variants or fragments of human Mcl-1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human Mcl-1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). Mcl-1 can be isolated from human, or may be produced recombinantly or by synthetic methods. 30 ME150999036v.1 132043-01120

[0101] The term "inhibit" or "inhibition" or “inhibiting,” as used herein, means to reduce a biologicalactivity or process by a measurable amount, and can include but does not require complete prevention or inhibition. In some embodiments, “inhibition” means to reduce the expression and / or activity of a Bcl-2 family protein (e.g., Bcl-2, Bcl-xL, Mcl-1) and / or one or more upstream modulators or downstream targets thereof.

[0102] The term “Mcl-1 inhibitor,” as used herein, refers to an agent capable of reducing theexpression 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. As used herein, the terms "derivative" and "analog" when referring to an Mcl-1 inhibitor, or the like, means any such compound that retains essentially the same, similar, or enhanced biological function or activity as compared to the original compound but has an altered chemical or biological structure.

[0103] As used herein, a “Mcl-1 inhibitor drug moiety”, “Mcl-1 inhibitor”, and the like refer to thecomponent 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.

[0104] The term “B-cell lymphoma-extra large” or “Bcl-xL,” as used herein, refers to any nativeform 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. The term also encompasses functional variants or fragments of human Bcl-xL, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human Bcl-xL (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). Bcl-xL can be isolated from human, or may be produced recombinantly or by synthetic methods.

[0105] The term “Bcl-xL inhibitor,” as used herein, refers to an agent capable of reducing theexpression and / or activity of Bcl-xL and / or one or more upstream modulators or downstream targets thereof. Exemplary Bcl-xL modulators (including exemplary inhibitors of Bcl-xL) are described in WO2010 / 080503, WO2010 / 080478, WO2013 / 055897, WO2013 / 055895, WO2016 / 094509, WO2016 / 094517, WO2016 / 094505, WO 2021 / 018858, WO 2021 / 018857, Tao et al., ACS Medicinal Chemistry Letters (2014), 5(10), 1088-109, and Wang et al., ACS Medicinal Chemistry Letters (2020), 11(10), 1829−1836, each of which are incorporated herein by reference as exemplary Bcl-xL 31 ME150999036v.1 132043-01120 modulators, including exemplary Bcl-xL inhibitors, that can be included as drug moieties in the disclosed ADCs.

[0106] As used herein, a “Bcl-xL inhibitor drug moiety”, “Bcl-xL inhibitor”, and the like refer to thecomponent 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.

[0107] The term “B-cell lymphoma 2” or “Bcl-2,” as used herein, refers to any native form of humanBcl-2, an anti-apoptotic member of the Bcl-2 protein family. The term encompasses full-length human Bcl-2 (e.g., UniProt Reference Sequence: P10415), as well as any form of human Bcl-2 that may result from cellular processing. The term also encompasses functional variants or fragments of human Bcl-2, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human Mcl-1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). Bcl-2 can be isolated from human, or may be produced recombinantly or by synthetic methods.

[0108] The term “Bcl-2 inhibitor,” as used herein, refers to an agent capable of reducing theexpression and / or activity of Bcl-2 and / or one or more upstream modulators or downstream targets thereof. Exemplary Bcl-2 modulators (including exemplary inhibitors of Bcl-2) 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.

[0109] As used herein, a “Bcl-2 inhibitor drug moiety”, “Bcl-2 inhibitor”, and the like refer to thecomponent 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.

[0110] The term “cancer,” as used herein, refers to the presence of cells possessing characteristicstypical 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. The term "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). Exemplary 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), 32 ME150999036v.1 132043-01120 chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. The terms “acute lymphoblastic leukemia” and “acute lymphocytic leukemia” can be used interchangeably to describe ALL. 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. In some embodiments, 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0111] As used herein, the term “tumor” refers to any mass of tissue that results from excessive cellgrowth or proliferation, either benign or malignant, including precancerous lesions. In some embodiments, 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.

[0112] The terms “tumor cell” and “cancer cell” may be used interchangeably herein and refer toindividual cells or the total population of cells derived from a tumor or cancer, including both non- tumorigenic cells and cancer stem cells. The terms “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.

[0113] The term “target-negative,” “target antigen-negative,” or “antigen-negative,” as used herein,refers to the absence of target antigen expression by a cell or tissue. The term “target-positive,” “target antigen-positive,” or “antigen-positive” refers to the presence of target antigen expression. For example, 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.

[0114] The terms “subject” and “patient” are used interchangeably herein to refer to any human ornon-human animal in need of treatment. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as any mammal. Non-limiting examples of mammals include humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, and guinea pigs. Non-limiting examples of non-mammals include birds and fish. In some embodiments, the subject is a human. 33 ME150999036v.1 132043-01120

[0115] The term “a subject in need of treatment,” as used herein, refers to a subject that wouldbenefit 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).

[0116] As used herein, the term “treat,” “treating,” or “treatment” refers to any improvement of anyconsequence 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, in addition to treating a subject having a disease, disorder, or condition, a composition disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of developing that disease, disorder, or condition.

[0117] As used herein, the term “prevent”, “preventing," or “prevention” of a disease, disorder, orcondition refers to the prophylactic treatment of the disease, disorder, or condition; or delaying the onset or progression of the disease, disorder, or condition.

[0118] As used herein, 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. 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.

[0119] As used herein, the terms "pharmaceutically acceptable carrier" and "physiologicallyacceptable carrier," which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered ADC compound or composition and / or any additional therapeutic agent in the composition. Pharmaceutically acceptable carriers may enhance or stabilize the composition or can 34 ME150999036v.1 132043-01120 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.

[0120] As used herein, the term "excipient" refers to an inert substance added to a pharmaceuticalcomposition 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. Other exemplary 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.

[0121] The term “pharmaceutically acceptable salt,” as used herein, refers to a salt which does notabrogate 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, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine. See, e.g., Haynes et al., “Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., “Pharmaceutical Salts,” J. Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated by reference herein.

[0122] In some embodiments, depending on their electronic charge, the antibody-drug conjugates(ADCs), linkers, payloads and linker-payloads described herein can contain a monovalent anionic counterion M1-. Any suitable anionic counterion can be used. In certain embodiments, the monovalent anionic counterion is a pharmaceutically acceptable monovalent anionic counterion. In certain embodiments, the monovalent anionic counterion M1- can be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, the monovalent anionic counterion M1- is trifluoroacetate or formate. 35 ME150999036v.1 132043-01120

[0123] As used herein, the term “therapeutically effective amount” or “therapeutically effectivedose,” 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). In some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects. In some embodiments, 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. In some embodiments, 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. In the case of cancer, 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.

[0124] As used herein, the term “prophylactically effective amount” or “prophylactically effectivedose,” 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. Typically, since 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. In some embodiments, a prophylactically effective amount can prevent the onset of disease symptoms, including symptoms associated with a cancer.

[0125] The term “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 antineoplastic payloads per antibody or antigen-binding fragment (Ab) in ADCs of Formula (I). In the present disclosure, one dual linker attaches two antineoplastic compounds (e.g. two BH3 mimetic drug moieties) to an antibody or antigen-binding fragment, 36 ME150999036v.1 132043-01120 therefore, p is 2 if the antibody or antigen-binding fragment only links with one dual linker having two antineoplastic compounds (e.g. two BH3 mimetic drug moieties) attached thereto. In compositions comprising multiple copies of ADCs of Formula (I), “average p” refers to the average number of antineoplastic compounds (e.g. two BH3 mimetic drug moieties) per antibody or antigen- binding fragment, also referred to as “average drug loading.” 1. Antibody-Drug Conjugates

[0126] The antibody-drug conjugate (ADC) compounds of the present disclosure include those withanti-cancer activity. In particular, the ADC compounds include an antibody or antigen-binding fragment conjugated (i.e., covalently attached by a dual linker) to two antineoplastic compounds as described herein. In some embodiments, the antineoplastic compound 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. Without being bound by theory, by targeting a Bcl-2 family protein (e.g., Mcl-1, Bcl-2 and / or Bcl-xL) expression and / or activity, in some embodiments, the ADCs disclosed herein may provide potent anti-cancer agents. Also, without being bound by theory, by conjugating the antineoplastic compound to an antibody that binds an antigen associated with expression in a tumor cell or cancer, the ADC may provide improved activity, better cytotoxic specificity, and / or reduced off-target killing as compared to the antineoplastic compound when administered alone.

[0127] In some embodiments, therefore, the components of the ADC are selected to (i) retain one ormore 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).

[0128] The ADC compounds of the present disclosure may selectively deliver an effective dose of acytotoxic or cytostatic agent to cancer cells or to tumor tissue. In some embodiments, the cytotoxic and / or cytostatic activity of the ADC is dependent on target antigen expression in a cell. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing a target 37 ME150999036v.1 132043-01120 antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit a cytotoxic and / or cytostatic effect on cancer cells that do not express a target antigen.

[0129] Provided herein, in certain aspects, are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) covalently linked to two antineoplastic payloads, such as a BH3 mimetic (D1and D2) through a dual linker (L). In some embodiments, for the ADC compounds provided herein, the antibody or antigen-binding fragment thereof (Ab) targets a cancer cell. In some embodiments, the antibody or antigen-binding fragment is able to bind to a tumor-associated antigen (e.g., CD74), e.g., with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell. In some embodiments, the ADCs internalize upon binding to a target cell, undergo degradation, and release the antineoplastic payload to kill cancer cells. The antineoplastic payloads, such as BH3 mimetics, may be released from the antibody and / or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0130] An exemplary ADC has the structure of Formula (I): wherein Ab = an antibody or antigen-binding fragment, L = a dual linker moiety, D1= anantineoplastic payload, such as a BH3 mimetic, D2 = an antineoplastic payload, such as a BH3mimetic, and a = the number of antineoplastic payloads, D1and D2, attached per antibody or antigen- binding fragment.

[0131] Below is a table showing exemplary dual linker-payload structures, wherein indicates thepoint of attachment to Ab. 38 ME150999036v.1 132043-01120 39 ME150999036v.1 132043-01120 A. Antibodies

[0132] The antibody or antigen-binding fragment (Ab) of Formula (I) includes within its scope ananti-CD74 antibody or antigen-binding fragment that specifically binds to a target antigen on a cell. In some embodiment, the antibody or antigen-binding fragment (Ab) of Formula (I) includes within its scope an anti-CD74 antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell (e.g., CD74). The antibody or antigen-binding fragment may bind to a target antigen 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. In some embodiments, the KD is 1 pM to 500 pM. In some embodiments, the KD is between 500 pM to 1 µM, 1 µM to 100 nM, or 100 mM to 10 nM.

[0133] In some embodiments, the antibody or antigen-binding fragment is a four-chain anti-CD74antibody (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 antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen- binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind a target cancer antigen and / or provide at least one function of the immunoglobulin.

[0134] In some embodiments, the antibody or antigen-binding fragment is an internalizing anti-CD74antibody or internalizing antigen-binding fragment thereof. In some embodiments, the internalizing anti-CD74 antibody or internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the surface of a cell and enters the cell upon binding. In some embodiments, the antineoplastic payload of the ADC is released from the anti-CD74 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 antigen-binding fragment, or by any other suitable release mechanism.

[0135] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments comprisemutations 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. In some embodiments, amino acid residues L234 and L235 of the IgG1 constant region are substituted to A234 and A235 (also known as “LALA”). In some embodiments, amino acid residue N297 of the IgG1 constant region is substituted to A297 (also known as “N297A”). In some embodiments, amino acid residues D265 and P329 of the IgG1 constant region are substituted to A265 and A329 (also known as “DAPA”). Other antibody Fc silencing mutations may also be used. In some embodiments, the Fc silencing mutations are used in combination, for example D265A, N297A and P329A (also known as “DANAPA”).

[0136] As set forth herein, if modifications are made to the antibodies or antigen-binding fragments,they are further designated with that modification. For example if select amino acids in the antibody have been changed to cysteines (e.g. E152C, S375C according to EU numbering of the antibody heavy chain to facilitate conjugation to linker-drug moieties) they are designated as “CysMab”; or if the 40 ME150999036v.1 132043-01120 antibody or antigen-binding fragment has been modified with Fc silencing mutations D265A, N297A and P329A of the IgG1 constant region according to EU numbering, “DANAPA” is added to the antibody name, or if the antibody or antigen-binding fragment has been modified with Fc silencing mutations D265A and P329A of the IgG1 constant region according to EU numbering, “DAPA” is added to the antibody name.

[0137] Amino acid sequences of exemplary antibodies or antigen-binding fragments of the presentdisclosure, in addition to exemplary antigen targets, are set forth in Tables A1-A3. Table A1. Antibodies Exemplified Table A2. Exemplary anti-CD74 VHmil x VK1aNQ Antibody Sequences 41 ME150999036v.1 132043-01120 Table A3. Amino acid sequences of VHmil x Vk1aNQ Fab fragments Table A4. Exemplary CD74 target antigen amino acid sequences

[0138] In some embodiments, the antibody or antigen-binding fragment of an ADC disclosed hereinmay 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. In some embodiments, 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 42 ME150999036v.1 132043-01120 KD of less than 1x10-8M) and 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.).

[0139] In some embodiments, the antibody or antigen-binding fragment of an ADC disclosed hereinfurther comprises human heavy and light chain constant domains or fragments thereof. For instance, the antibody or antigen-binding fragment of the described ADCs may comprise a human IgG heavy chain constant domain (such as an IgG1) and a human kappa or lambda light chain constant domain. In some embodiments, the antibody or antigen-binding fragment of the described ADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain with a human Ig kappa light chain constant domain.

[0140] In some embodiments, the target antigen for an ADC of the present disclosure is CD74. Insome embodiments, the anti-CD74 antibody or antigen-binding fragment thereof is a VHmil x VK1aNQ antibody or antigen-binding fragment thereof.

[0141] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesthree heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:12.

[0142] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesthree heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:4, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15.

[0143] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesthree heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:6, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:7, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:8; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15.

[0144] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesthree heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:9, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15. 43 ME150999036v.1 132043-01120

[0145] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesa heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:18, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 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:17 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:18.

[0146] In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprisesthe 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:20 or a sequence that is at least 95% identical to SEQ ID NO:20. In some embodiments, the anti-CD74 antibody or antigen- binding fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof 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:20.

[0147] Residues in two or more polypeptides are said to "correspond" if the residues occupy ananalogous 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.

[0148] In some embodiments, amino acid substitutions are of single residues. Insertions usually willbe 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 B. 44 ME150999036v.1 132043-01120 Table B Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0149] In some embodiments where variant antibody sequences are used in an ADC, the variantstypically 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. Alternatively, the variant may be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed.

[0150] In some embodiments, while the disclosed linkers and antineoplastic payloads, such as BH3mimetics, are surprisingly effective with several different tumor-targeting antibodies, CD74-targeting antibodies such as VHmil x VK1aNQ, 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. B. Linkers 45 ME150999036v.1 132043-01120

[0151] In some embodiments, the linker in an ADC is stable extracellularly in a sufficient manner tobe therapeutically effective. In some embodiments, 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).

[0152] As used herein, “stable,” in the context of a linker or ADC comprising a linker, means that nomore 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. In some embodiments, 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. In some embodiments, 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.

[0153] Whether a linker is stable extracellularly can be determined, for example, by including anADC 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. In some embodiments, 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. Thus, 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.

[0154] Linkers may impact the physico-chemical properties of an ADC. As many cytotoxic agentsare hydrophobic in nature, linking them to the antibody with an additional hydrophobic moiety may lead to aggregation. ADC aggregates are insoluble and often limit achievable drug loading onto the antibody, which can negatively affect the potency of the ADC. Protein aggregates of biologics, in general, have also been linked to increased immunogenicity. As shown below, linkers disclosed herein result in ADCs with low aggregation levels and desirable levels of drug loading.

[0155] A linker may be "cleavable" or "non-cleavable" (Ducry and Stump (2010) BioconjugateChem. 21:5-13). Cleavable linkers are designed to release the drug moiety (e.g., antineoplastic payloads, such as BH3 mimetics, topoisomerase 1 inhibitors, or anti-mitotic drugs) when subjected to 46 ME150999036v.1 132043-01120 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.

[0156] As used herein, when a partial structure of a compound is illustrated, a wavy line ( )indicates the point of attachment of the partial structure to the rest of the molecule.

[0157] Dual linkers of the present disclosure are described in International PCT ApplicationPCT / US2023 / 022990, which is incorporated herein by reference in its entirety. In some embodiments, the dual linker is selected from: (L17), and 47 ME150999036v.1 132043-01120 (L19). wherein each A1or A2independently is a bond, -OC(=O)-* , wherein * indicates the point of attachment to D1or D2; indicates the point of attachment to the Ab; and indicates the point of direct attachment to D1or D2. C. Drug Moieties

[0158] In some embodiments, an intermediate, which is the precursor of the linker moiety, is reactedwith the drug moiety (e.g., BH3 mimetics, such as a Mcl-1 inhibitor, a Bcl-2 inhibitor and / or a Bcl-xL inhibitor) under appropriate conditions. In some embodiments, 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 (drug plus linker), 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. Alternatively, 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.

[0159] A number of different reactions are available for covalent attachment of the drug moietyand / or linker moiety to the antibody or antigen-binding fragment. This is often accomplished by reaction of one or more amino acid residues of the antibody or antigen-binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of the aromatic amino acids. For instance, 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. Additionally, 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. Also available for attachment of drugs (e.g., a BH3 mimetic) to binding agents is the 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. See International PCT 48 ME150999036v.1 132043-01120 Application PCT / US2023 / 022990, section of Drug Moieties, which is incorporated herein by reference in its entirety. Other techniques are known to the skilled artisan and within the scope of the present disclosure. The drug moieties of present disclosure are listed in Table C below. Table C 49 ME150999036v.1 132043-01120 Drug Loading

[0160] Drug loading is represented by p (or 2a in ADCs of formula (I) of the present disclosure), andis 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. In some embodiments, a is an integer from 1 to 16. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0161] 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. For example, 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). 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).

[0162] In some embodiments, the number of drug moieties that can be conjugated to an antibody orantigen-binding fragment is limited by the number of free cysteine residues. For example, where the attachment is a cysteine thiol group, 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. Generally, 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 50 ME150999036v.1 132043-01120 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. Therefore, 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. In some embodiments, an optimal ratio may be 2, 4, 6, or 8. In some embodiments, an optimal ratio may be 2 or 4.

[0163] In some embodiments, an antibody or antigen-binding fragment is exposed to reducingconditions 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. 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). In embodiments, 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)). In some embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as cysteine.

[0164] The drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting themolar 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.

[0165] In some embodiments, free cysteine residues are introduced into the amino acid sequence ofthe antibody or antigen-binding fragment. For example, 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. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a "ThioFab." Similarly, 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 51 ME150999036v.1 132043-01120 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. (2012) Methods Enzymol. 502:123-38). In some embodiments, 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.

[0166] Where more than one nucleophilic group reacts with a drug-linker intermediate or a linkermoiety reagent followed by drug moiety reagent, in a reaction mixture comprising multiple copies of the antibody or antigen-binding fragment and linker moiety, then 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. In some embodiments, 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) 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). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, 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.

[0167] In some embodiments, the average number of drug moieties per antibody or antigen-bindingfragment is about 2. In some embodiments, 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.

[0168] In some embodiments, the average number of drug moieties per antibody or antigen-bindingfragment 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, 52 ME150999036v.1 132043-01120 about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 4.

[0169] In some embodiments, the term “about,” as used with respect to the average number of drugmoieties 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.

[0170] Individual ADC compounds, or “species,” may be identified in the mixture by massspectroscopy and separated by, e.g., UPLC or HPLC, e.g. hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, 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.

[0171] In some embodiments, higher drug loading (e.g., p > 16) may cause aggregation, insolubility,toxicity, or loss of cellular permeability of certain antibody-drug conjugates. Higher drug loading may also negatively affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p < 2) may reduce the potency of certain ADCs against target- expressing cells. In some embodiments, 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.

[0172] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. A drug loading and / or an average drug loading of more than about 16 may also affect stability of the ADC, due to loss of one or more chemical bonds required to stabilize the antibody or antigen-binding fragment.

[0173] The present disclosure includes methods of producing the described ADCs. Briefly, theADCs 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. In some embodiments, the ADCs can be prepared using a linker having reactive functionalities for covalently attaching to the drug moiety and to the antibody or antigen- 53 ME150999036v.1 132043-01120 binding fragment. In some embodiments, the antibody or antigen-binding fragment is functionalized to prepare a functional group that is reactive with a linker or a drug-linker intermediate. For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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. The generation of the ADCs can be accomplished by techniques known to the skilled artisan.

[0174] In some embodiments, an ADC is produced by contacting an antibody or antigen-bindingfragment 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. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting the drug moiety. In other embodiments, 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. In other embodiments, 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. In some embodiments, 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.

[0175] The ADCs prepared according to the methods described above may be subjected to apurification step. The purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge or isoelectric point-based chromatography, mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof. 2. Therapeutic Uses and Compositions 54 ME150999036v.1 132043-01120

[0176] Disclosed herein are methods of using the compositions described herein, e.g., the disclosedADC compounds and compositions, in treating a subject for a disorder, e.g., a cancer. Compositions, e.g., 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.

[0177] Methods of determining whether an ADC exerts a cytostatic and / or cytotoxic effect on a cellare known. For example, 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). Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.

[0178] For determining cytotoxicity, necrosis or apoptosis (programmed cell death) may bemeasured. 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).

[0179] Apoptosis may also be determined by measuring morphological changes in a cell. Forexample, as with necrosis, 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 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. Apoptosis may also be determined, in some embodiments, by screening for caspase activity. In some embodiments, a Caspase-Glo® Assay can be used to measure activity of caspase-3 and caspase-7. In some embodiments, the assay provides a luminogenic caspase-3 / 7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, 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. In some embodiments, 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 55 ME150999036v.1 132043-01120 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.

[0180] Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such asneutral red, trypan blue, Crystal Violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such an assay, 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.

[0181] Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolicallyactive cells. In some embodiments, 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. In this assay, in some embodiments, 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.

[0182] Cell viability may also be measured, e.g., by measuring the reduction of tetrazolium salts. Insome embodiments, 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) may be assessed using an MTT cell viability assay, as described in the examples provided herein. In this assay, in some embodiments, 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.

[0183] In certain aspects, the present disclosure features a method of killing, inhibiting or modulatingthe 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. 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. In some embodiments, 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- 56 ME150999036v.1 132043-01120 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0184] In some embodiments, the disclosed ADCs may be administered in any cell or tissue thatexpresses CD74, such as a CD74-expressing cancer cell or tissue. An exemplary embodiment includes a method of killing a CD74-expressing cancer cell or tissue. The method may be used with any cell or tissue that expresses CD74, such as a cancerous cell or a metastatic lesion. Non-limiting examples of CD74-expressing cancers include a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, 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, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the CD74-expressing cancer is a hematological cancer. In some embodiments, the CD74-expressing 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). In some embodiments, the CD74-expressing cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0185] 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. For example, cells that express CD74 (e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells), can be cultured in vitro in culture medium and the contacting step can be affected by adding the ADC to the culture medium. The method will result in killing of cells expressing CD74, including in particular cancer cells expressing CD74. Alternatively, 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. This approach can be used for antibodies targeting cell surface antigens (e.g., CD74).

[0186] The in vivo effect of a disclosed ADC therapeutic composition can be evaluated in a suitableanimal model. For example, 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. 57 ME150999036v.1 132043-01120

[0187] In vivo assays that evaluate the promotion of tumor death by mechanisms such as apoptosismay also be used. In some embodiments, 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.

[0188] Further provided herein are methods of treating a disorder, e.g., a cancer. The compositionsdescribed 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., CD74) (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.

[0189] An exemplary embodiment is a method of treating a subject having or suspected of having acancer, 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). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, 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. In some embodiments, the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

[0190] Another exemplary embodiment is a method of delivering antineoplastic payloads (e.g. BH3mimetics) to a cell expressing CD74, comprising conjugating the antineoplastic payloads (e.g. BH3 mimetics) to an antibody that immunospecifically binds to a CD74 epitope and exposing the cell to the ADC. Exemplary cancer cells that express CD74 for which the ADCs of the present disclosure are indicated include hematological cancer cells.

[0191] In certain aspects, the present disclosure further provides methods of reducing or inhibitinggrowth of a tumor (e.g., a CD74-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC. In some embodiments, 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. In some embodiments, the tumor is 58 ME150999036v.1 132043-01120 resistant or refractory to treatment with the antibody or antigen-binding fragment of the ADC (e.g., an anti-CD74 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.

[0192] An exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in asubject, 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). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, 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, or spleen cancer. In some embodiments, 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.

[0193]

[0194] Another exemplary embodiment is a method of delaying or slowing the growth of a tumor ina 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). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is CD74.

[0195] In some embodiments, the tumor is a breast cancer, gastric cancer, bladder cancer, braincancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, 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.

[0196] An exemplary embodiment is a method of reducing or inhibiting the growth of ahematological 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). In some embodiments, the hematological cancer expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, 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 59 ME150999036v.1 132043-01120 leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non-Hodgkin's lymphoma or myelodysplasia syndrome (MDS). In some embodiments, 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.

[0197] In certain aspects, the present disclosure further provides methods of reducing or slowing theexpansion of a cancer cell population (e.g., a CD74-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC.

[0198] An exemplary embodiment is a method of reducing or slowing the expansion of a cancer cellpopulation 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). In some embodiments, the cancer cell population expresses a target antigen. In some embodiments, the target antigen is CD74. In some embodiments, the cancer cell population is from a tumor or a hematological cancer. In some embodiments, 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, or head and neck cancer. In some embodiments, the cancer cell population is from acute myeloid leukemia, multiple myeloma, or B-cell lymphoma. In some embodiments, 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. In some embodiments, 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.

[0199] Also provided herein are methods of determining whether a subject having or suspected ofhaving a cancer will be responsive to treatment with the disclosed ADCs and compositions. 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 60 ME150999036v.1 132043-01120 (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. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. In some embodiments, 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.).

[0200] Further provided herein are therapeutic uses of the disclosed ADCs and compositions. Anexemplary 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 CD74-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 CD74-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., a CD74-expressing cancer). Methods for identifying subjects having cancers that express a target antigen (e.g., CD74) are known in the art and may be used to identify suitable patients for treatment with a disclosed ADC compound or composition.

[0201] Moreover, ADCs of the present disclosure may be administered to a non-human mammalexpressing 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).

[0202] The therapeutic compositions used in the practice of the foregoing methods may beformulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier suitable for the desired delivery method. An exemplary embodiment is 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). 61 ME150999036v.1 132043-01120

[0203] 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. In many cases, 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.

[0204] A pharmaceutical composition of the present disclosure can be administered by a variety ofmethods 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. Potentially effective routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous), intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. In some embodiments, 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). Depending on the route of administration, 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). Administration can be either systemic or local.

[0205] The therapeutic compositions disclosed herein may be sterile and stable under the conditionsof 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. In some embodiments, 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. In some embodiments, 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.

[0206] Typically, a therapeutically effective amount or efficacious amount of a disclosedcomposition, e.g., a disclosed ADC, is employed in the pharmaceutical compositions of the present 62 ME150999036v.1 132043-01120 disclosure. The composition, e.g., one comprising an ADC, 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.

[0207] Dosage regimens for compositions disclosed herein, e.g., those comprising ADCs alone or incombination 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). For example, 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. In some embodiments, treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration. In some embodiments, the ADC is administered to the patient daily, weekly, monthly, or any time period in between. For any particular subject, 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.

[0208] Dosage values for compositions comprising an ADC and / or any additional therapeuticagent(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. In general, 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. 63 ME150999036v.1 132043-01120

[0209] Toxicity and therapeutic efficacy of compounds provided herein can be determined bystandard 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. For example, the compositions and methods disclosed herein may initially be evaluated in xenogeneic cancer models (e.g., an NCI-H929 multiple myeloma mouse model).

[0210] In some embodiments, an ADC or composition comprising an ADC is administered on asingle 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. In therapeutic applications, 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.

[0211] The above therapeutic approaches can be combined with any one of a wide variety ofadditional surgical, chemotherapy, or radiation therapy regimens. In some embodiments, 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.

[0212] Kits for use in the therapeutic and / or diagnostic applications described herein are alsoprovided. 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. Directions and or other information may also be included on an insert(s) or label(s), which is included with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate 64 ME150999036v.1 132043-01120 that an ADC or composition within the kit is used for diagnosing or treating a condition, such as a cancer a described herein.

[0213] In some embodiments, a kit comprises an ADC or composition comprising an ADC. In someembodiments, 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. Instructions for use can include guidance for therapeutic applications including suggested dosages and / or modes of administration, e.g., in a patient having or suspected of having a cancer. In some embodiments, the kit comprises an ADC and instructions for use of the ADC in treating, preventing, and / or diagnosing a cancer.

[0214] It is known that elevated expression levels of BCL2 family members (e.g., Bcl-xL) correlateswith resistance to radiation therapy and chemotherapy. Antibody-drug conjugates (ADCs) 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. Without wishing to be bound by theory, it is believed that 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. In some embodiments, antibody drug conjugates described herein, are administered to a subject having cancer in an amount effective to sensitize the tumor cells. As used herein, 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

[0215] In some embodiments, the present disclosure provides methods of treatment wherein theantibody-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.

[0216] In certain embodiments, a combination described herein comprises a PD-1 inhibitor. In someembodiments, the PD-1 inhibitor is chosen from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In some embodiments, the PD-1 inhibitor is PDR001. PDR001 is also known as Spartalizumab.

[0217] In certain embodiments, a combination described herein comprises a LAG-3 inhibitor. Insome embodiments, the LAG-3 inhibitor is chosen from LAG525 (Novartis), BMS-986016 (Bristol- Myers Squibb), or TSR-033 (Tesaro). 65 ME150999036v.1 132043-01120

[0218] In certain embodiments, a combination described herein comprises a TIM-3 inhibitor. Insome embodiments, the TIM-3 inhibitor is MBG453 (Novartis), TSR-022 (Tesaro), LY-3321367 (Eli Lily), Sym23 (Symphogen), BGB-A425 (Beigene), INCAGN-2390 (Agenus), BMS-986258 (BMS), RO-7121661 (Roche), or LY-3415244 (Eli Lilly).

[0219] In certain embodiments, a combination described herein comprises a PDL1 inhibitor. In oneembodiment, the PDL1 inhibitor is chosen from FAZ053 (Novartis), atezolizumab (Genentech), durvalumab (Astra Zeneca), or avelumab (Pfizer).

[0220] In certain embodiments, a combination described herein comprises a GITR agonist. In someembodiments, the GITR agonist is chosen from GWN323 (NVS), BMS-986156, MK-4166 or MK- 1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (Incyte / Agenus), AMG 228 (Amgen) or INBRX-110 (Inhibrx).

[0221] In some embodiments, a combination described herein comprises an IAP inhibitor. Insome embodiments, the IAP inhibitor comprises LCL161 or a compound disclosed in International Application Publication No. WO 2008 / 016893.

[0222] In an embodiment, the combination comprises an mTOR inhibitor, e.g., RAD001 (alsoknown as everolimus).

[0223] In an embodiment, the combination comprises a HDAC inhibitor, e.g., LBH589. LBH589 isalso known as panobinostat.

[0224] In an embodiment, the combination comprises an IL-17 inhibitor, e.g., CJM112.

[0225] In certain embodiments, a combination described herein comprises an estrogen receptor (ER)antagonist. In some embodiments, the estrogen receptor antagonist is used in combination with a PD- 1 inhibitor, a CDK4 / 6 inhibitor, or both. In some embodiments, the combination is used to treat an ER positive (ER+) cancer or a breast cancer (e.g., an ER+ breast cancer).

[0226] In some embodiments, the estrogen receptor antagonist is a selective estrogen receptordegrader (SERD). SERDs are estrogen receptor antagonists which bind to the receptor and result in e.g., degradation or down-regulation of the receptor (Boer K. et al., (2017) Therapeutic Advances in Medical Oncology 9(7): 465-479). ER is a hormone-activated transcription factor important for e.g., the growth, development and physiology of the human reproductive system. ER is activated by, e.g., the hormone estrogen (17beta estradiol). ER expression and signaling is implicated in cancers (e.g., breast cancer), e.g., ER positive (ER+) breast cancer. In some embodiments, the SERD is chosen from LSZ102, fulvestrant, brilanestrant, or elacestrant.

[0227] In some embodiments, the SERD comprises a compound disclosed in InternationalApplication Publication No. WO 2014 / 130310, which is hereby incorporated by reference in its entirety.

[0228] In some embodiments, the SERD comprises LSZ102. LSZ102 has the chemical name: (E)-3-(4-((2-(2-(1,1-difluoroethyl)-4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)phenyl)acrylic 66 ME150999036v.1 132043-01120 acid. In some embodiments, the SERD comprises fulvestrant (CAS Registry Number: 129453-61-8), or a compound disclosed in International Application Publication No. WO 2001 / 051056, which is hereby incorporated by reference in its entirety. In some embodiments, the SERD comprises elacestrant (CAS Registry Number: 722533-56-4), or a compound disclosed in U.S. Patent No. 7,612,114, which is incorporated by reference in its entirety. Elacestrant is also known as RAD1901, ER-306323 or (6R)-6-{2-[Ethyl({4-[2-(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}- 5,6,7,8-tetrahydronaphthalen-2-ol. Elacestrant is an orally bioavailable, non-steroidal combined selective estrogens receptor modulator (SERM) and a SERD. Elacestrant is also disclosed, e.g., in Garner F et al., (2015) Anticancer Drugs 26(9):948-56. In some embodiments, the SERD is brilanestrant (CAS Registry Number: 1365888-06-7), or a compound disclosed in International Application Publication No. WO 2015 / 136017, which is incorporated by reference in its entirety.

[0229] In some embodiments, the SERD is chosen from RU 58668, GW7604, AZD9496,bazedoxifene, pipendoxifene, arzoxifene, OP-1074, or acolbifene, e.g., as disclosed in McDonell et al. (2015) Journal of Medicinal Chemistry 58(12) 4883-4887.

[0230] Other exemplary estrogen receptor antagonists are disclosed, e.g., in WO 2011 / 156518, WO2011 / 159769, WO 2012 / 037410, WO 2012 / 037411, and US 2012 / 0071535, all of which are hereby incorporated by reference in their entirety

[0231] In certain embodiments, a combination described herein comprises an inhibitor of Cyclin-Dependent Kinases 4 or 6 (CDK4 / 6). In some embodiments, the CDK4 / 6 inhibitor is used in combination with a PD-1 inhibitor, an estrogen receptor (ER) antagonist, or both. In some embodiments, the combination is used to treat an ER positive (ER+) cancer or a breast cancer (e.g., an ER+ breast cancer). In some embodiments, the CDK4 / 6 inhibitor is chosen from ribociclib, abemaciclib (Eli Lilly), or palbociclib.

[0232] In some embodiments, the CDK4 / 6 inhibitor comprises ribociclib (CAS Registry Number:1211441-98-3), or a compound disclosed in U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entirety.

[0233] In some embodiments, the CDK4 / 6 inhibitor comprises a compound disclosed in InternationalApplication Publication No. WO 2010 / 020675 and U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entirety.

[0234] In some embodiments, the CDK4 / 6 inhibitor comprises ribociclib (CAS Registry Number:1211441-98-3). Ribociclib is also known as LEE011, KISQALI®, or 7-cyclopentyl-N,N-dimethyl-2- ((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide.

[0235] In some embodiments, the CDK4 / 6 inhibitor comprises abemaciclib (CAS Registry Number:1231929-97-7). Abemaciclib is also known as LY835219 or N-[5-[(4-Ethyl-1-piperazinyl)methyl]-2- pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]-2- pyrimidinamine. Abemaciclib is a CDK inhibitor selective for CDK4 and CDK6 and is disclosed, e.g., in Torres-Guzman R et al. (2017) Oncotarget 10.18632 / oncotarget.17778. 67 ME150999036v.1 132043-01120

[0236] In some embodiments, the CDK4 / 6 inhibitor comprises palbociclib (CAS Registry Number:571190-30-2). Palbociclib is also known as PD-0332991, IBRANCE® or 6-Acetyl-8-cyclopentyl-5- methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one. Palbociclib inhibits CDK4 with an IC50 of 11nM, and inhibits CDK6 with an IC50 of 16nM, and is disclosed, e.g., in Finn et al. (2009) Breast Cancer Research 11(5):R77.

[0237] In certain embodiments, a combination described herein comprises an inhibitor of chemokine(C-X-C motif) receptor 2 (CXCR2). In some embodiments, the CXCR2 inhibitor is chosen from 6- chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxy-N-methoxy-N- methylbenzenesulfonamide, danirixin, reparixin, or navarixin.

[0238] In some embodiments, the CSF-1 / 1R binding agent is chosen from an inhibitor ofmacrophage colony-stimulating factor (M-CSF), e.g., a monoclonal antibody or Fab to M-CSF (e.g., MCS110), a CSF-1R tyrosine kinase inhibitor (e.g., 4-((2-(((1R,2R)-2- hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide or BLZ945), a receptor tyrosine kinase inhibitor (RTK) (e.g., pexidartinib), or an antibody targeting CSF-1R (e.g., emactuzumab or FPA008). In some embodiments, the CSF-1 / 1R inhibitor is BLZ945. In some embodiments, the CSF-1 / 1R binding agent is MCS110. In other embodiments, the CSF-1 / 1R binding agent is pexidartinib.

[0239] In certain embodiments, a combination described herein comprises a c-MET inhibitor. c-MET, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays key roles in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of c- MET may induce cell death in tumor cells overexpressing c-MET protein or expressing constitutively activated c-MET protein. In some embodiments, the c-MET inhibitor is chosen from capmatinib (INC280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib, tivantinib, or golvatinib.

[0240] In certain embodiments, a combination described herein comprises a transforming growthfactor beta (also known as TGF-β TGFβ, TGFb, or TGF-beta, used interchangeably herein) inhibitor. In some embodiments, the TGF-β inhibitor is chosen from fresolimumab or XOMA 089.

[0241] In certain embodiments, a combination described herein comprises an adenosine A2a receptor(A2aR) antagonist (e.g., an inhibitor of A2aR pathway, e.g., an adenosine inhibitor, e.g., an inhibitor of A2aR or CD-73). In some embodiments, the A2aR antagonist is used in combination with a PD-1 inhibitor, and one or more (e.g., two, three, four, five, or all) of a CXCR2 inhibitor, a CSF-1 / 1R binding agent, LAG-3 inhibitor, a GITR agonist, a c-MET inhibitor, or an IDO inhibitor. In some embodiments, the combination is used to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma (e.g., a refractory melanoma). In some embodiments, the A2aR antagonist is chosen from PBF509 (NIR178) (Palobiofarma / Novartis), CPI444 / V81444 (Corvus / Genentech), AZD4635 / HTL-1071 (AstraZeneca / Heptares), Vipadenant (Redox / Juno), GBV-2034 (Globavir), AB928 (Arcus Biosciences), Theophylline, Istradefylline (Kyowa Hakko Kogyo), Tozadenant / SYN- 115 (Acorda), KW-6356 (Kyowa Hakko Kogyo), ST-4206 (Leadiant Biosciences), or 68 ME150999036v.1 132043-01120 Preladenant / SCH 420814 (Merck / Schering). Without wishing to be bound by theory, it is believed that in some embodiments, inhibition of A2aR leads to upregulation of IL-1b.

[0242] In certain embodiments, a combination described herein comprises an inhibitor ofindoleamine 2,3-dioxygenase (IDO) and / or tryptophan 2,3-dioxygenase (TDO). In some embodiments, the IDO inhibitor is used in combination with a PD-1 inhibitor, and one or more (e.g., two, three, four, or all) of a TGF-β inhibitor, an A2aR antagonist, a CSF-1 / 1R binding agent, a c-MET inhibitor, or a GITR agonist. In some embodiments, the combination is used to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma (e.g., a refractory melanoma). In some embodiments, the IDO inhibitor is chosen from (4E)-4-[(3-chloro-4-fluoroanilino)- nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as epacadostat or INCB24360), indoximod (NLG8189), (1-methyl-D-tryptophan), α-cyclohexyl-5H-Imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919), indoximod, BMS-986205 (formerly F001287).

[0243] In certain embodiments, a combination described herein comprises a Galectin, e.g., Galectin-1 or Galectin-3, inhibitor. In some embodiments, the combination comprises a Galectin-1 inhibitor and a Galectin-3 inhibitor. In some embodiments, the combination comprises a bispecific inhibitor (e.g., a bispecific antibody molecule) targeting both Galectin-1 and Galectin-3. In some embodiments, the Galectin inhibitor is used in combination with one or more therapeutic agents described herein. In some embodiments, the Galectin inhibitor is chosen from an anti-Galectin antibody molecule, GR-MD-02 (Galectin Therapeutics), Galectin-3C (Mandal Med), Anginex, or OTX-008 (OncoEthix, Merck).

[0244] In some embodiments, a combination described herein comprises an inhibitor of the MAPkinase pathway including ERK inhibitors, MEK inhibitors and RAF inhibitors.

[0245] In some embodiments, a combination described herein comprises a MEK inhibitor. In someembodiments, the MEK inhibitor is chosen from Trametinib, selumetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, or G02443714.

[0246] In some embodiments, the MEK inhibitor is trametinib. Trametinib is also known as JTP-74057, TMT212, N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo- 3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide, or Mekinist (CAS Number 871700-17-3).

[0247] In some embodiments, the MEK inhibitor comprises selumetinib which has the chemicalname: (5-[(4-bromo-2-chlorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H- benzimidazole-6-carboxamide. Selumetinib is also known as AZD6244 or ARRY 142886, e.g., as described in PCT Publication No. WO2003077914.

[0248] In some embodiments, the MEK inhibitor comprises AS703026, BIX 02189 or BIX 02188.

[0249] In some embodiments, the MEK inhibitor comprises 2-[(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI-1040 or PD184352), e.g., as described in PCT Publication No. WO2000035436). 69 ME150999036v.1 132043-01120

[0250] In some embodiments, the MEK inhibitor comprises N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]- benzamide (also known as PD0325901), e.g., as described in PCT Publication No. WO2002006213).

[0251] In some embodiments, the MEK inhibitor comprises 2’-amino-3’-methoxyflavone (alsoknown as PD98059) which is available from Biaffin GmbH & Co., KG, Germany.

[0252] In some embodiments, the MEK inhibitor comprises 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126), e.g., as described in US Patent No. 2,779,780).

[0253] In some embodiments, the MEK inhibitor comprises XL-518 (also known as GDC-0973)which has a CAS No. 1029872-29-4 and is available from ACC Corp.

[0254] In some embodiments, the MEK inhibitor comprises G-38963.

[0255] In some embodiments, the MEK inhibitor comprises G02443714 (also known as AS703206)

[0256] Additional examples of MEK inhibitors are disclosed in WO 2013 / 019906, WO 03 / 077914,WO 2005 / 121142, WO 2007 / 04415, WO 2008 / 024725 and WO 2009 / 085983, the contents of which are incorporated herein by reference. Further examples of MEK inhibitors include, but are not limited to, 2,3-Bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126 and described in US Patent No. 2,779,780); (3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8,9,16-trihydroxy- 3,4-dimethyl-3,4,9, 19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] (also known as E6201, described in PCT Publication No. WO2003076424); vemurafenib (PLX-4032, CAS 918504- 65-1); (R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3- d]pyrimidine-4,7(3H,8H)-dione (TAK-733, CAS 1035555-63-5); pimasertib (AS-703026, CAS 1204531-26-9); 2-(2-Fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6- dihydropyridine-3-carboxamide (AZD 8330); and 3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-N- (2-hydroxyethoxy)-5-[(3-oxo-[1,2]oxazinan-2-yl)methyl]benzamide (CH 4987655 or Ro 4987655).

[0257] In some embodiments, a combination described herein comprises a RAF inhibitor.

[0258] RAF inhibitors include, but are not limited to, Vemurafenib (or Zelboraf®, PLX-4032, CAS918504-65-1), GDC-0879, PLX-4720 (available from Symansis), Dabrafenib (or GSK2118436), LGX 818, CEP-32496, UI-152, RAF 265, Regorafenib (BAY 73-4506), CCT239065, or Sorafenib (or Sorafenib Tosylate, or Nexavar®).

[0259] In some embodiments, the RAF inhibitor is Dabrafenib.

[0260] In some embodiments, the RAF inhibitor is LXH254.

[0261] In some embodiments, a combination described herein comprises an ERK inhibitor.

[0262] ERK inhibitors include, but are not limited to, LTT462, ulixertinib (BVD-523), LY3214996,GDC-0994, KO-947 and MK-8353.

[0263] In some embodiments, the ERK inhibitor is LTT462. LTT462 is 4-(3-amino-6-((1S,3S,4S)-3-fluoro-4-hydroxy¬cyclohexyl)pyrazin-2-yl)-N-((S)-1-(3-bromo-5-fluorophenyl)-2- (methylamino)¬ethyl)-2-fluorobenzamide and is the compound of the following structure: 70 ME150999036v.1 132043-01120

[0264] The preparation of LTT462 is described in PCT patent application publicationWO2015 / 066188. LTT462 is an inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK 1 / 2).

[0265] In some embodiments, a combination described herein comprises a taxane, a vinca alkaloid, aMEK inhibitor, an ERK inhibitor, or a RAF inhibitor.

[0266] In some embodiments, a combination described herein comprises at least two inhibitorsselected, independently, from a MEK inhibitor, an ERK inhibitor, and a RAF inhibitor.

[0267] In some embodiments, a combination described herein comprises an anti-mitotic drug.

[0268] In some embodiments, a combination described herein comprises a taxane.

[0269] Taxanes include, but are not limited to, docetaxel, paclitaxel, or cabazitaxel. In someembodiments, the taxane is docetaxel.

[0270] In some embodiments, a combination described herein comprises a vinca alkaloid.

[0271] Vinca alkaloids include, but are not limited to, vincristine, vinblastine, and leurosine.

[0272] In some embodiments, a combination described herein comprises a topoisomerase inhibitor.

[0273] 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.

[0274] In one embodiment, a combination described herein includes an interleukin-1 beta (IL-1β)inhibitor. In some embodiments, the IL-1β inhibitor is chosen from canakinumab, gevokizumab, Anakinra, or Rilonacept.

[0275] In certain embodiments, a combination described herein comprises an IL-15 / IL-15Racomplex. In some embodiments, the IL-15 / IL-15Ra complex is chosen from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune).

[0276] In certain embodiments, a combination described herein comprises a mouse double minute 2homolog (MDM2) inhibitor. The human homolog of MDM2 is also known as HDM2. In some embodiments, an MDM2 inhibitor described herein is also known as a HDM2 inhibitor. In some embodiments, the MDM2 inhibitor is chosen from HDM201 or CGM097. 71 ME150999036v.1 132043-01120

[0277] In an embodiment the MDM2 inhibitor comprises (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)- 1,2-dihydroisoquinolin-3(4H)-one (also known as CGM097) or a compound disclosed in PCT Publication No. WO 2011 / 076786 to treat a disorder, e.g., a disorder described herein). In one embodiment, a therapeutic agent disclosed herein is used in combination with CGM097.

[0278] In some embodiments, a combination described herein comprises a hypomethylating agent(HMA). In some embodiments, the HMA is chosen from decitabine or azacitidine.

[0279] In some embodiments, a combination described herein comprises a glucocorticoid. In someembodiments, the glucocorticoid is dexamethasone.

[0280] In some embodiments, a combination described herein comprises asparaginase.

[0281] In certain embodiments, a combination described herein comprises an inhibitor acting on anypro-survival proteins of the Bcl2 family. In certain embodiments, a combination described herein comprises a Bcl-2 inhibitor. In some embodiments, the Bcl-2 inhibitor is venetoclax (also known as ABT-199):

[0282] In one embodiment, the Bcl-2 inhibitor is selected from the compounds described in WO2013 / 110890 and WO 2015 / 011400. In some embodiments, 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). In some embodiments, 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: (compound A1). 72 ME150999036v.1 132043-01120

[0283] In some embodiments, 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).

[0284] In one embodiment, the antibody-drug conjugates or combinations disclosed herein aresuitable for the treatment of cancer in vivo. For example, 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. For example, to achieve antigen-specific enhancement of immunity, the combination can be administered together with an antigen of interest. A combination disclosed herein can be administered in either order or simultaneously.

[0285] Provided are also protocols for some aspects of analytical methodology for evaluatingantibody conjugates of the invention. Such analytical methodology and results can demonstrate that the 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. In general, it is desirable to have higher amounts of monomer and lower amounts of, for example, aggregated antibody due to the impact of, for example, aggregates on other properties of the antibody sample such as but not limited to clearance rate, immunogenicity, and toxicity. A further example is the determination of the hydrophobicity by hydrophobic interaction chromatography (HIC) wherein the hydrophobicity of a sample is assessed relative to a set of standard antibodies of known properties. In general, it is desirable to have low hydrophobicity due to the impact of hydrophobicity on other properties of the antibody sample such as but not limited to 73 ME150999036v.1 132043-01120 aggregation, aggregation over time, adherence to surfaces, hepatotoxicity, clearance rates, and pharmacokinetic exposure. See Damle, N.K., Nat Biotechnol. 2008; 26(8):884-885; Singh, S.K., Pharm Res. 2015; 32(11):3541-71. When measured by hydrophobic interaction chromatography, higher hydrophobicity index scores (i.e. elution from HIC column faster) reflect lower hydrophobicity of the conjugates. As shown in Examples below, a majority of the tested antibody conjugates showed a hydrophobicity index of greater than 0.8. In some embodiments, provided are antibody conjugates having a hydrophobicity index of 0.8 or greater, as determined by hydrophobic interaction chromatography. EXAMPLES

[0286] The following examples provide illustrative embodiments of the disclosure. One of ordinaryskill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The examples provided do not in any way limit the disclosure.

[0287] Abbreviations:AcOH acid acetic aq. aqueous Boc tert-butyloxycarbonyl Boc2O di-tert-butyl dicarbonate DCC: dicyclohexylcarbodiimide DCE: dichloroethane DCM dichloromethane DIEA / DIPEA N,N-Diisopropylethylamine DIAD diisopropylazodicarboxylate DMAP 4-dimethylaminopyridine DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride DMF dimethyl formamide DMSO dimethyl sulfoxide EDC / EDC-HCl 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc(AcOEt) ethyl acetate EtOH ethanol FMOC fluorenylmethoxycarbonyl HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate 74 ME150999036v.1 132043-01120 HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOAt 1-Hydroxy-7-azabenzotriazole HFIP hexafluoroisopropanol IPA, i-PrOH or 2-PrOH isopropyl alcohol JosiPhos Pd G3 {(R)-1-[(Sp)-2-(dicyclohexylphosphino)ferrocenyl]ethyldi-tert- butylphosphine}[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate MeCN acetonitrile MeOH methanol MTBE methyl tert-butyl ether or tert-butyl methyl ether NaOtBu sodium tert-butoxide NMP N-methylpyrrolidine TES triethylsilane TBAF tetrabutyl ammonium fluoride TBAI tetrabutyl ammonium iodide TBTU [Bis(dimethylamino)methylene]-1H-benzotriazolium 3-Oxide Tetrafluoroborate Pd(AtaPhos)2Cl2bis(di-tert-butyl(4-dimethylaminophenyl)phosphine) dichloropalladium(II) PdCl2(PPh3)2dichlorobis(triphenylphosphine)palladium(II) Pd2(dba)3tris(dibenzylideneacetone)dipalladium(0) PMB para-methoxybenzyl pTsOH para-toluene sulfonic acid PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate TSTU O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate THF tetrahydrofuran TFA trifluoroacetic acid TFE 2,2,2-Trifluoroethanol TBAF tetrabutylammonium fluoride TBDMSCl tert-butyldimethylsilyl chloride TEA trimethylamine rt room temperature sat. saturated 75 ME150999036v.1 132043-01120 Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene Example 1. Synthesis and Characterization of Payload and Precursors Thereof

[0288] Exemplary payloads and precursors thereof were synthesized using exemplary methodsdescribed in this example.

[0289] a. Material, Methods & General Procedure for the preparation of the payloads P1-P4:

[0290] All reagents obtained from commercial sources were used without further purification.Anhydrous solvents were obtained from commercial sources and used without further drying.

[0291] Payload P1 was prepared according to the method described in Example 30 of InternationalPCT publication WO2015 / 097123, which is incorporated herein by reference. Below is a table showing structures of payloads P1-P4. 76 ME150999036v.1 132043-01120

[0292] Column Chromatography

[0293] Automated flash column chromatography was performed on ISCO CombiFlash® Rf 200 orCombiFlash®Rf+ LumenTMusing RediSep®Rf Normal-phase Silica Flash Columns (35-70µm, 60 Å), RediSep Rf Gold®Normal-phase Silica High Performance Columns (20-40µm, 60 Å), RediSep®Rf Reversed-phase C18 Columns (40-63 µm, 60 Å), or RediSep Rf Gold®Reversed-phase C18 High Performance Columns (20-40 µm, 100 Å).

[0294] TLC

[0295] Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60F254 silica-gel.

[0296] Microwave Reactions

[0297] Microwave heating was performed with a CEM Discover® SP, or with an Anton PaarMonowave Microwave Reactor.

[0298] NMR

[0299] 1H-NMR measurements were performed on a Bruker Avance III 500 MHz spectrometer, aBruker Avance III 400 MHz spectrometer, or a Bruker DPX-400 spectrometer using DMSO-d6 or CDCl3 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-d6 and 7.26 ppm for CDCl3) 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). 77 ME150999036v.1 132043-01120

[0300] Analytical LC-MS

[0301] Certain compounds of the present invention were characterized by high performance liquidchromatography-mass spectroscopy (HPLC-MS) on Agilent HP1200 with Agilent 6140 quadrupole LC / MS, operating in positive or negative ion electrospray ionisation mode. Molecular weight scan range is 100 to 1350. Parallel UV detection was done at 210 nm and 254 nm. Samples were supplied as a 1 mM solution in ACN, or in THF / H2O (1:1) with 5 µL loop injection. LCMS analyses were performed on two instruments, one of which was operated with basic, and the other with acidic eluents.

[0302] Basic LCMS: Gemini-NX, 3 µm, C18, 50 mm × 3.00 mm i.d. column at 23 °C, at a flow rateof 1 mL min-1 using 5 mM ammonium bicarbonate (Solvent A) and acetonitrile (Solvent B) with a gradient starting from 100% Solvent A and finishing at 100% Solvent B over various / certain duration of time.

[0303] Acidic LCMS: KINATEX XB-C18-100A, 2.6 ^m, 50 mm*2.1 mm column at 40 °C, at aflow 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.

[0304] Certain other compounds of the present invention were characterized HPLC-MS underspecific 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. Gradient elutions were run by defining flow rates and percentage mixtures of the following mobile phases, using HPLC-grade solvents:

[0305] Solvent A: 10 mM aqueous ammonium formate + 0.04% (v / v) formic acid

[0306] Solvent B: Acetonitrile + 5.3% (v / v) Solvent A + 0.04% (v / v) formic acid.

[0307] Retention times (RT) for these named methods are reported in minutes. Ionisation is recordedin positive mode, negative mode, or positive-negative switching mode. Specific details for individual methods follow.

[0308] LCMS-V-B methods

[0309] Using an Agilent 1200 SL series instrument linked to an Agilent MSD 6140 singlequadrupole 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 µm, C18, 50 mm x 2.1 mm at 55 ºC. Gradient details for methods LCMS-V-B1 and LCMS-V-B2: 78 ME150999036v.1 132043-01120

[0310] LCMS-V-C method

[0311] Using an Agilent 1200 SL series instrument linked to an Agilent MSD 6140 singlequadrupole with an ESI-APCI multimode source; column: Agilent Zorbax Eclipse plus 3.5 µm, C18(2), 30 mm x 2.1 mm at 35 ºC. Gradient details for method LCMS-V-C:

[0312] Preparative HPLC

[0313] Certain compounds of the present invention were purified by high performance liquidchromatography (HPLC) on an Armen Spot Liquid Chromatography or Teledyne EZ system with a Gemini-NX® 10 µM C18, 250 mm × 50 mm i.d. column running at a flow rate of 118 mL min-1 with UV diode array detection (210 – 400 nm) using 25 mM aqueous NH4HCO3 solution and MeCN or 0.1% TFA in water and MeCN as eluents.

[0314] Certain other compounds of the present invention were purified by HPLC under specificnamed methods as follows:

[0315] HPLC-V-A methods

[0316] These were performed on a Waters FractionLynx MS autopurification system, with aGemini®5 µm C18(2), 100 mm × 20 mm i.d. column from Phenomenex, running at a flow rate of 20 cm3min-1with UV diode array detection (210–400 nm) and mass-directed collection. 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.

[0317] Method HPLC-V-A1 (pH 4):79 ME150999036v.1 132043-01120

[0318] Solvent A: 10 mM aqueous ammonium acetate + 0.08% (v / v) formic acid; Solvent B:acetonitrile + 5% (v / v) Solvent A + 0.08% (v / v) formic acid

[0319] Method HPLC-V-A2 (pH 9):

[0320] Solvent A: 10 mM aqueous ammonium acetate + 0.08% (v / v) conc. ammonia; Solvent B:acetonitrile + 5% (v / v) Solvent A + 0.08% (v / v) conc. ammonia

[0321] HPLC-V-B methods

[0322] Performed on an AccQPrep HP125 (Teledyne ISCO) system, with a Gemini® NX 5 µmC18(2), 150 mm × 21.2 mm i.d. column from Phenomenex, running at a flow rate of 20 cm3min-1with UV (214 and 254 nm) and ELS detection.

[0323] Method HPLC-V-B1 (pH 4):

[0324] Solvent A: water + 0.08% (v / v) formic acid; solvent B: acetonitrile + 0.08% (v / v) formic acid.

[0325] Method HPLC-V-B2 (pH 9):

[0326] Solvent A: water + 0.08% (v / v) conc. ammonia; solvent B: acetonitrile + 0.08% (v / v) conc.ammonia.

[0327] Method HPLC-V-B3 (neutral):

[0328] Solvent A: water; Solvent B: acetonitrile.

[0329] Analytical GC-MS

[0330] Combination gas chromatography and low resolution mass spectrometry (GC-MS) wasperformed on Agilent 6850 gas chromatograph and Agilent 5975C mass spectrometer using 15 m × 0.25 mm column with 0.25 µm HP-5MS coating and helium as carrier gas. Ion source: EI+, 70 eV, 230°C, quadrupole: 150°C, interface: 300°C.

[0331] High-resolution MS

[0332] High-resolution mass spectra were acquired on an Agilent 6230 time-of-flight massspectrometer equipped with a Jet Stream electrospray ion source in positive ion mode. Injections of 0.5μl were directed 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.

[0333] Chemical naming

[0334] IUPAC-preferred names were generated using ChemAxon’s ‘Structure to Name’ (s2n)functionality within MarvinSketch or JChem for Excel (JChem versions 16.6.13 – 18.22.3), or with the chemical naming functionality provided by Biovia® Draw 4.2.

[0335] General Procedures

[0336] General procedure 1a-Buchwald80 ME150999036v.1 132043-01120

[0337] The appropriate aryl bromide (1 eq.), the appropriate aniline (1.1 eq.), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (0.04 eq.) and NaOtBu (2 eq.) were suspended in toluene (3 mL / mmol aryl bromide). The mixture was sparged with N2 and then Pd2(dba)3 (0.04 eq.) was added, and the mixture was heated for 1 h at 110°C under microwave irradiation or at 70°C under a N2 atmosphere until complete conversion was observed. The mixture was allowed to cool to rt, diluted with water and filtered through a celite cartridge. The filtrate was partitioned between EtOAc and water. The phases were separated, and the aqueous phase was extracted with EtOAc. The combined organic phases were dried over MgSO4 and concentrated in vacuo. The crude material was purified by automated flash chromatography using heptane and EtOAc as eluents.

[0338] General procedure 1b - Buchwald

[0339] To a solution of the appropriate aryl bromide (1 eq.) and the appropriate aniline (1 eq.) inTHF (4.5 mL / mmol aryl bromide) was added NaOtBu (1 eq.) and chloro(2-di-t-butylphosphino- 2',4',6'-tri-i-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl] palladium(II) [t-BuXPhos Palladacycle Gen. 1] (0.04 eq.) and the mixture was stirred at rt under N2until complete conversion was observed. The mixture was diluted with water and the organic layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4and concentrated in vacuo. The crude material was purified by automated flash chromatography using heptane or MeOH and EtOAc as eluents.

[0340] General procedure 2a – Amide coupling

[0341] To a solution of the appropriate acid (1 eq.) in DCE (12 mL / mmol acid) was added 4Åmolecular sieves followed by 1-chloro-N,N,2-trimethyl-1-propenylamine (2 eq.) and then the mixture was stirred under N2at rt for 30 mins. A solution of the appropriate aniline (1.5 eq.) and pyridine (2 eq.) in DCE (2.5 mL / mmol aniline) was added and the mixture stirred at 80°C under N2for 18 h. The mixture was allowed to cool to rt and partitioned between DCM and water. The phases were separated, and the organic phase was washed with sat. aq. NaHCO3solution, dried over MgSO4and concentrated in vacuo. The crude material was purified by automated flash chromatography using heptane and EtOAc or DCM and MeOH as eluents.

[0342] General procedure 2b – Amide coupling

[0343] To a solution of the appropriate acid (1 eq.) in DCM (12 mL / mmol acid) was added oxalyldichloride (1.7 eq.) and then the mixture was stirred under N2 at rt for 30 mins. The solution was concentrated in vacuo at rt then the appropriate aniline (1.2 eq.) in DCE (12 mL / mmol acid) was added and the mixture was stirred at 80°C under N2 for 4-18 h. The reaction mixture was concentrated in vacuo and the crude material was purified by automated flash chromatography using DCM and MeOH as eluents.

[0344] General procedure 4a – Amide coupling

[0345] To a solution of the appropriate amine (1 eq.) in DMF (6 mL / mmol amine) was added theappropriate carboxylic acid (1.5 eq.) followed by DIPEA (2 eq.) and HATU (1 eq.) and the mixture 81 ME150999036v.1 132043-01120 was stirred at rt until complete conversion was observed. The reaction mixture was partitioned between DCM and water. The phases were separated and the organic phase dried over MgSO4 and concentrated. Purification by automated flash chromatography using DCM and MeOH as eluents was followed by preparative HPLC automated flash chromatography at pH 9 or pH 4, using water and MeCN as eluents.

[0346] General procedure 7a ^ O-Alkylation

[0347] A stirred suspension of the appropriate phenol (1 eq.) and K2CO3 (5 eq.) in MeCN (5-10mL / mmol) was heated at 70°C for 20 min. The appropriate alkyl halide (1.6 eq.) was added, and heating continued until complete conversion was observed. The mixture was allowed to cool to rt andfiltered, washing with EtOAc. The filtrate was concentrated in vacuo and the residue was purified byautomated flash chromatography using heptane and EtOAc or DCM and MeOH as eluents.

[0348] General procedure 8 ^ Ester hydrolysis

[0349] A solution of the appropriate ester (1 eq.) in MeOH (3-11 mL / mmol) was treated with 2 M aq.NaOH solution (2 eq.) and stirred at rt for 24 h. The MeOH was removed in vacuo and the aqueous residue was neutralised with 2 M aq. HCl solution and then purified by reverse phase automated flash chromatography using water and MeCN as eluents.

[0350] Preparation I: 2-benzyloxycarbonyl-6-(4-ethoxycarbonyl-1,5-dimethyl-pyrrol-2-yl)-3,4-dihydro-1H-isoquinoline-7-carboxylic acid

[0351] The synthesis of the title compound is described in WO2015 / 011164 A1, in Example 805Step B.

[0352] Preparation IIa: 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 82 ME150999036v.1 132043-01120

[0353] Step A: (3R)-3-methyl-1,2,3,4-tetrahydroisoquinoline hydrochloride

[0354] The synthesis of the title compound is described in WO2015 / 011164 A1 at Preparation 2b.

[0355] 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

[0356] To a solution of Preparation I (3 g, 6.3 mmol, 1 eq.) and the product from Step A (1.27 g, 6.93mmol, 1.1 eq.) in DMF (30 mL) was added DIPEA (3.13 mL, 18.89 mmol, 3 eq.) and PyBop (3.6 g, 6.93 mmol, 1.1 eq.) and the mixture stirred at rt for 12 h. 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. NaHCO3 solution and water, dried over MgSO4 and concentrated in vacuo to afford the title product (3.7 g, 6.11 mmol, 97%).

[0357] HRMS calcd for C H N O : 605.289, foun +37 39 3 5 d 606.296 [M+H]

[0358] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.52 – 6.75 (m, 11H), 6.43 – 6.00 (m, 1H), 5.39 –1.95 (m, 21H), 1.31 – 0.49 (m, 6H).

[0359] 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 83 ME150999036v.1 132043-01120

[0360] To a solution of the product from Step B (3.7 g, 6.11 mmol, 1 eq.) in MeOH (70 mL) andEtOH (20 mL) was added 10% Pd / C (100 mg). The mixture was evacuated and backfilled with N2, then evacuated and flushed with H2 and then shaken at rt for 6 h under an atmosphere of H2. The mixture was filtered through a celite cartridge, washing with EtOH. The solvent was removed in vacuo to afford the title product (2.94 g, quant.).

[0361] HRMS calcd for C29H33N3O3: 471.252, found 472.259 [M+H]+

[0362] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.24 – 6.81 (m, 6H), 6.40 – 5.99 (m, 1H), 5.34 – 1.93(m, 19H), 1.30 – 0.52 (m, 7H).

[0363] 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

[0364] To a solution of the product from Step C (2.94 g, 6.23 mmol, 1 eq.) in THF (45 mL) andwater (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. The solvents were removed in vacuo and the residue partitioned between EtOAc and sat aq. NH4Cl solution. The phases were separated, and the organic phase was washed with sat. aq. NaCl solution, dried over MgSO4 and concentrated in vacuo to afford the title product (3.39 g, 5.93 mmol, 95%).

[0365] HRMS calcd for C34H41N3O5: 571.305, found 572.315 [M+H]+

[0366] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.33 – 6.79 (m, 6H), 6.41 – 6.00 (m, 1H), 5.34 – 1.99(m, 19H), 1.51 – 1.37 (m, 9H), 1.28 – 0.52 (m, 6H). 84 ME150999036v.1 132043-01120

[0367] 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

[0368] LiOH.H2O (995 mg, 23.72 mmol, 4 eq.) was added to a solution of the product from Step D(3.39 g, 5.93 mmol, 1 eq.) in a mixture of MeOH (40 mL) and water (20 mL) and heated at 100°C for 24 h. The mixture was allowed to cool to rt and MeOH removed in vacuo. The aqueous residue was acidified with 1 M aq. HCl solution to pH 5, diluted with water (10 mL) and extracted with DCM. The combined organic extracts were dried over MgSO4 and concentrated in vacuo. Purification by automated flash chromatography eluting with a gradient of 0 - 6% MeOH in DCM afforded the title product (3.1 g, 5.7 mmol, 96%).

[0369] HRMS calcd for C32H37N3O5: 543.273, found 544.283 [M+H]+

[0370] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.53 (s, 1H), 7.32 – 6.80 (m, 6H), 6.38 – 6.06 (m,1H), 5.37 – 1.89 (m, 17H), 1.50 – 1.38 (m, 9H), 1.08 – 0.50 (m, 3H).

[0371] Preparation IIIa: 4-[(tert-butyldimethylsilyl)oxy]aniline

[0372] To a solution of imidazole (13.72 g, 201.6 mmol, 2.5 eq.) and 4-aminophenol (8.8 g, 80.64mmol, 1 eq.) in MeCN (150 mL), cooled to 0°C under N2, was added TBDMSCl (12.76 g, 84.67 mmol, 1.05 eq.). The mixture was allowed to warm to rt and stirred for 4 h. The mixture was partitioned between EtOAc and water. The phases were separated, and the organic phase was washed with brine, dried over MgSO4and concentrated in vacuo. The residue was diluted with heptane (40 mL) and left to stir for 1 h. The precipitate was removed by filtration and the filtrate concentrated in vacuo to afford the title product (19.6 g, quant.).

[0373] HRMS calcd for (C12H21NOSi): 223.139, found 224.162 [M+H]+

[0374] 1H NMR (400 MHz, DMSO-d6) δ ppm: 6.56 – 6.48 (m, 2H), 6.48 – 6.40 (m, 2H), 4.87 (br s,2H), 0.92 (s, 9H), 0.10 (s, 6H).

[0375] Preparation IVa: 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 85 ME150999036v.1 132043-01120

[0376] Step A: 4-bromo-1,5-dimethylpyrrole-2-carbonitrile

[0377] A solution of Br2 (6.74 mL, 131.08 mmol, 1.05 eq.) in AcOH (70 mL) was added dropwise toa solution of 1,5-dimethylpyrrole-2-carbonitrile (15 g, 124.84 mmol, 1 eq.) in AcOH (250 mL), cooled to 10°C. The mixture was stirred at 10°C for 1 h, and then allowed to warm to rt, and again stirred for 3 h. The mixture was poured into ice water (500 mL) and the resulting precipitate was collected by filtration, washed with water, and dried in vacuo to afford the title product, (23.7 g, 119.07 mmol, 95%).

[0378] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.05 (s, 1H), 3.65 (s, 3H), 2.22 (s, 3H).

[0379] Step B: 4-({4-[(tert-butyldimethylsilyl)oxy]phenyl}amino)-1,5-dimethylpyrrole-2-carbonitrile

[0380] Using General procedure 1a and the product from Step A (6.1 g, 30.65 mmol, 1 eq.) as theappropriate aryl bromide and Preparation IIIa (7.19 g, 32.18 mmol, 1.05 eq.) as the appropriate aniline afforded the title product (8.2 g, 24 mmol, 78%).

[0381] HRMS calcd for C19H27N3OSi: 341.192, found 342.207 [M+H]+

[0382] 1H NMR (400 MHz, DMSO-d6) δ ppm: 6.87 (s, 1H), 6.73 (s, 1H), 6.64 – 6.56 (m, 2H), 6.54– 6.46 (m, 2H), 3.61 (s, 3H), 2.09 (s, 3H), 0.92 (s, 9H), 0.12 (s, 6H). 86 ME150999036v.1 132043-01120

[0383] 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

[0384] 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, 9.2 mmol, 2 eq.) as the appropriate aniline. Purification by automated flash chromatography eluting with a gradient of 0 - 6% MeOH in DCM afforded the title product (2.49 g, 2.87 mmol, 62%).

[0385] HRMS calcd for C51H62N6O5Si: 866.455, found 867.465 [M+H]+

[0386] 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

[0387] A solution of the product from Step C (2.8 g, 3.23 mmol, 1 eq.) in MeOH (5 mL) was treatedwith 3 M HCl in MeOH (10 mL, 30 mmol) and stirred at rt for 3 h. The solvents were removed in vacuo and then dried under high vacuum to afford the title product (2.53 g, quant.).

[0388] HRMS calcd for C40H40N6O3: 652.316, found 653.326 [M+H]+

[0389] Preparation Va: {4-[2-(morpholin-4-yl)ethoxy]phenyl}acetic acid

[0390] Step A: methyl 2-{4-[2-(morpholin-4-yl)ethoxy]phenyl}acetate 87 ME150999036v.1 132043-01120

[0391] Using General procedure 7a and methyl 4-hydroxyphenylacetate (2.5 g, 15.04 mmol, 1 eq.) asthe appropriate phenol and 4-(2-chloroethyl)morpholine hydrochloride (4.48 g, 24.07 mmol, 1.6 eq.) as the appropriate alkyl halide afforded the title product (3.24 g, 11.59 mmol, 77%).

[0392] LRMS calcd for (C15H21NO4): 279.2, found 280.2 [M+H]+

[0393] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.20 – 7.12 (m, 2H), 6.93 – 6.85 (m, 2H), 4.06 (t, J =5.8 Hz, 2H), 3.60 (s, 3H), 3.59 (s, 2H), 3.59 – 3.56 (m, 4H), 2.68 (t, J = 5.8 Hz, 2H), 2.50 – 2.43 (m, 4H).

[0394] Step B: {4-[2-(morpholin-4-yl)ethoxy]phenyl}acetic acid

[0395] The product from Step A (3.26 g, 11.67 mmol, 1 eq.) was hydrolysed using General procedure8 to afford the title product (3.1 g, 11.63 mmol, quant).

[0396] LRMS calcd for (C14H19NO4): 265.1, found 266.2 [M+H]+

[0397] 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.15 – 7.09 (m, 2H), 6.87 – 6.79 (m, 2H), 4.04 (t, J =5.8 Hz, 2H), 3.60 – 3.54 (m, 4H), 3.34 (s, 2H), 2.66 (t, J = 5.8 Hz, 2H), 2.49 – 2.42 (m, 4H).

[0398] Preparation VIa: 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

[0399] 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 88 ME150999036v.1 132043-01120

[0400] A solution of Preparation IIa (3 g, 5.52 mmol, 1 eq.) in MeOH (20 mL) was treated with 3 MHCl 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.).

[0401] LRMS calcd for C27H29N3O3: 443.2, found 444.4 [M+H]+

[0402] 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.79 – 9.40 (br m, 2H), 7.42 – 6.79 (m, 6H), 6.43 –6.04 (m, 1H), 5.37 – 1.87 (m, 17H), 1.14 – 0.46 (m, 3H).

[0403] 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

[0404] To a solution of Preparation Va (1 g, 3.77 mmol, 1 eq) in anhydrous DCM (10 mL) under N2,was slowly added 2 M oxalyl chloride solution in DCM (0.43 mL, 4.52 mmol, 1.2 eq.) followed by DMF (1 drop), and the mixture stirred for 1 h. The solvents were removed in vacuo, the acid chloride intermediate was dissolved in anhydrous DCM (7 mL) and added dropwise to a stirred solution of the product from Step A (1.63 g, 3.39 mmol, 0.9 eq.) and DIPEA (2.5 mL, 15.08 mmol, 4 eq.) in anhydrous DCM (10 mL) under N2. The mixture was stirred at rt for 2 h, then quenched with MeOH (5 mL) and concentrated in vacuo. Purification by automated flash chromatography eluting with a gradient of 0 - 8% MeOH in DCM afforded the title product (1.2 g, 1.74 mmol, 46%).

[0405] LRMS calcd for (C41H46N4O6): 690.3, found 691.6 [M+H]+

[0406] 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.53 (s, 1H), 7.32 – 6.78 (m, 10H), 6.39 – 6.05 (m,1H), 5.35 – 1.88 (m, 31H), 1.11 – 0.48 (m, 3H).

[0407] Preparation VIIb: 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

[0408] Step A: 1,2-dimethyl-5-[7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-1,2,3,4-tetrahydroisoquinolin-6-yl]pyrrole-3-carboxylic acid 89 ME150999036v.1 132043-01120

[0409] To a solution of Preparation I (45.9 g, 96.3 mmol, 1 eq.) in DMF (240 mL) was added TBTU(34.1 g, 106 mmol, 1.1 eq.) followed by TEA (40.5 mL, 291 mmol, 3 eq.). 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. The product was extracted with DCM / IPA=3 / 1 (3 × 400 mL). The organic phase was dried over MgSO4 and concentrated in vacuo. The residue was triturated in diethyl ether / acetonitrile. The formed yellow powder was filtered out then dried in vacuo to afford the title product (38.51 g, 86.8 mmol, 90%).

[0410] 1H NMR (500 MHz, DMSO-d6) δ ppm 7.35-6.8 (m, 6 H), 6.35 / 6.28 / 6.13 (s / s / s, 1 H), 5.34-3.23 (m, 6 H), 5.01 / 4.88 / 3.78 (m / m / m, 1 H), 3.46 / 3.43 / 3.38 / 3.15 (s / s / s / s, 3 H), 3.12-2.98 (m, 2 H), 3.02-2.05 (m, 2 H), 2.49 / 2.41 / 1.96 (s / s / s, 3 H), 1.03 / 0.85 / 0.74 / 0.56 (d / d / d / d, 3 H);13C NMR (125 MHz, DMSO-d6) δ ppm 168.9 / 168.4 / 168.2, 166.5 / 166.3 / 166.2, 111.3 / 111.2 / 110.8, 48.3 / 48.1 / 43 / 42.2, 34.4 / 34.2 / 34 / 32.6, 32.2 / 32.1 / 31.9, 25.4, 18.6 / 16.8 / 16.4, 11.8 / 11.7 / 11.2.

[0411] HRMS-ESI (m / z) [M+H]+ calcd for C27H30N3O3: 444.2282, found 444.2267

[0412] 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

[0413] To the biphasic mixture of the product from Step A (13.96 g, 28.33 mmol, 1 eq.) dissolved indioxane (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 eq.) was added dropwise and the mixture was stirred at rt for 24 h. To the reaction mixture 2M aq. HCl (42.5 mL; 85 mmol, 3 eq.) was added dropwise then after stirring for 10 min it was extracted with DCM. The phases were separated, and the 90 ME150999036v.1 132043-01120 organic phase was washed with sat. aq. NaCl solution, dried over MgSO4 and concentrated in vacuo. The crude material was purified by automated flash chromatography using DCM and EtOAc as eluents affording the title product (14.77 g, 22.2 mmol, 78%).

[0414] 1H NMR (500 MHz, DMSO-d6) δ ppm 11.18 (br., 1 H), 7.95-7.26 (br., 8 H), 7.26-6.8 (br., 6H), 6.52-6.06 (br., 1 H), 5.09-3.74 (br., 3 H), 4.54 (br., 2 H), 4.47 (d, 2 H), 4.31 (t, 1 H), 3.59 (br., 2 H), 3.5-3.19 (br., 3 H), 2.8 (t, 2 H), 2.55-1.93 (br., 3 H), 2.45 (br., 2 H), 1.2-0.41 (br., 3 H);13C NMR (125 MHz, DMSO-d6) δ ppm 111.1, 67.2, 47.5, 45.7, 41.6, 34.6, 32, 28.3, 16.8, 11.6.

[0415] HRMS-ESI (m / z) [M+H]+ calcd for C42H40N3O5: 666.2962, found 666.2961

[0416] Step C: 9H-fluoren-9-ylmethyl 6-[4-[[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-(5-cyano-1,2-dimethyl-pyrrol-3-yl)carbamoyl]-1,5-dimethyl-pyrrol-2-yl]-7-[(3R)-3-methyl-3,4-dihydro-1H- isoquinoline-2-carbonyl]-3,4-dihydro-1H-isoquinoline-2-carboxylate

[0417] Using General procedure 2b and the product from Step B (10.67 g, 16.03 mmol, 1 eq.) as theappropriate acid, oxalyl dichloride (2.17 mL, 25.6 mmol, 1.6 eq.) and the product from Step B of Preparation IVa (6.57 g, 19.2 mmol, 1.2 eq.) as the appropriate aniline afforded the title product (8.12 g, 8.21 mmol, 51%).

[0418] 1H NMR (500 MHz, DMSO-d6) δ ppm 8.05-6.43 (m, 19 H), 5.7-4.95 (s, 1 H), 5.12-2.26 (m,14 H), 3.71-3 (s, 6 H), 2.48-1.75 (s, 6 H), 1.13-0.44 (d, 3 H), 0.92-0.77 (s, 9 H), 0.15-0.05 (s, 6 H).

[0419] HRMS-ESI (m / z) [M+H]+ calcd for C61H65N6O5Si: 989.4780, found 989.4779

[0420] 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

[0421] 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. The reaction mixture was concentrated in vacuo and the residue was partitioned between DCM and water. The phases were separated, and the organic phase was washed with sat. aq. NaCl solution, dried over MgSO4 and concentrated in vacuo. The crude material was purified by automated flash chromatography using DCM and MeOH as eluents to afford the title product (4.51 g, 5.88 mmol, 72%). 91 ME150999036v.1 132043-01120

[0422] 1H NMR (500 MHz, DMSO-d6) δ ppm 7.36-6.35 (m, 11 H), 5.55-4.91 (s, 1 H), 5.37-1.97 (m,12 H), 3.72-2.96 (s, 6 H), 2.48-1.75 (s, 6 H), 1.16-0.39 (d, 3 H), 1-0.76 (s, 9 H), 0.17-0 (s, 6 H).

[0423] HRMS-ESI (m / z) [M+H]+ calcd for C46H55N6O3Si: 767.4099, found 767.4103

[0424] Preparation of P2: 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

[0425] Step 1: Ethyl 1,2-dimethyl-1H-pyrrole-3-carboxylate92 ME150999036v.1 132043-01120

[0426] To a solution of ethyl 2-methyl-1H-pyrrole-3-carboxylate (10 g, 65.3 mmol) and methyliodide (8.95 mL, 130.6 mmol) in 70 mL of dimethylformamide cooled at 0°C is added, in three portions, sodium hydride 60 % w / w (2.6 g, 65.3 mmol). The reaction mixture is stirred at 0°C for 1 hour. Then, the 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. The organic phase is dried over MgSO4, filtered, concentrated to dryness and purified by chromatography over silica gel (petroleum ether / AcOEt gradient).

[0427] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 6.65 (d, 1H); 6.3 (1d, 1H); 4.1 (1q, 2H); 3.5 (s, 3H);2.4 (s, 3H); 1.5 (1t, 3H).

[0428] Step 2: Ethyl 5-(5-chloro-2-formylphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylate

[0429] 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 MgSO4and 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.

[0430] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 9.8 (s, 1H); 7.91-7.69-7.61 (d, 3H); 6.5 (s, 1H); 4.2(q, 2H); 3.4 (s, 3H); 2.55 (s, 3H); 1.28 (t, 3H).

[0431] Step 3: 4-Chloro-2-[4-(ethoxycarbonyl)-1,5-dimethyl-1H-pyrrol-2-yl]benzoic acid

[0432] A solution is prepared containing the compound obtained in Step 2 (12.8 g, 42 mmol) and 2-methyl-2-butene (35.7 mL, 336 mmol) in a mixture containing 20 mL of acetone and 20 mL of tetrahydrofuran. There are added, dropwise, 200 mL of an aqueous solution containing a mixture of sodium chlorite (NaClO2, 13.3 g, 147 mmol) and sodium hydrogen phosphate (NaHPO4, 14.5 g, 105 mmol). 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 MgSO4and 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.

[0433] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 13 (m, 1H); 7.85-7.41(m, 3H); 6.3 (s, 1H); 4.15 (q,2H); 3.25 (s, 3H); 2.5 (s, 3H); 1.25 (t, 3H).

[0434] Step 4: {(3S)-2-[(4-Methylphenyl)sulphonyl]-1,2,3,4-tetrahydroisoquinolin-3-yl}methyl 4-methylbenzenesulphonate 93 ME150999036v.1 132043-01120

[0435] 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.

[0436] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.75 (d, 2H); 7.6 (d, 2H); 7.5 (d, 2H); 7.3 (d, 2H);7.15-6.9 (m, 4H); 4.4-4.15 (dd, 2H); 4.25 (m, 1H); 4.0-3.8 (2dd, 2H); 2.7 (2dd, 2H); 2.45 (s, 3H); 2.35 (s, 3H).

[0437] Step 5: (3S)-3-(iodomethyl)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinoline

[0438] To a suspension of the compound obtained in Step 4 (4 g, 8.5 mmol) in acetonitrile (10 mL) isadded sodium iodide (1.4 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. The crude is purified by chromatography over silica gel using cyclohexane and ethyl acetate as eluants, the expected product is obtained as a white powder.

[0439] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.64 (d, 2H), 7.28 (d, 2H), 7.15-7 (m, 4H), 4.5-4.3(2d, 2H), 4.14 (m, 1H), 3.22 (m, 2H), 2.82 (m, 2H), 2.31 (s, 3H).

[0440] MS (ESI): m / z 427 [M]+.

[0441] Step 6: diethyl 2-[[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]methyl]propanedioate

[0442] To a suspension of sodium hydride (442 mg, 11 mmol) in THF (8 mL) is added dropwisediethyl 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. After cooling, the reaction mixture is poured into saturated aqueous ammonium chloride solution, extracted three times with dichloromethane. The organic phase is washed with brine, dried over MgSO4 and concentrated to dryness. The crude is purified by chromatography over silica gel using cyclohexane and ethyl acetate as eluants, affording the desired compound.

[0443] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.6 (d, 2H), 7.3 (d, 2H), 7.1 (m, 4H), 4.6 / 4.25 (2d,2H), 4.2 (m, 1H), 4.1 (m, 4H), 3.55 (t, 1H), 2.6 (m, 2H), 2.3 (s, 3H), 1.85 (2m, 2H), 1.2 (m, 6H).

[0444] Step 7: 3-[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]propanoic acid94 ME150999036v.1 132043-01120

[0445] To a solution of the compound obtained in Step 6 (1 g, 2.2 mmol) in a mixture ethanol (8 mL)and water (5 mL) is added lithium hydroxide monohydrate (0.23 g, 5.4 mmol). The reaction mixture is heated to 85°C overnight. After cooling, the ethanol is evaporated. The aqueous reaction mixture is diluted with water (10 mL) and a solution of 1M HCl is added to reach pH=3. The reaction mixture is extracted with ethyl acetate twice. 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 MgSO4 and concentrated to dryness, affording the desired compound.

[0446] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 12.15 (m, 1H), 7.65 (d, 2H), 7.3 (d, 2H), 7.15-7 (2m,4H), 4.6 / 4.29 (2d, 2H), 4.2 (m, 1H), 2.6 (2dd, 2H), 2.35 (s, 3H), 2.25 (t, 2H), 1.52 (quad, 2 H).

[0447] Step 8: N,N-dimethyl-3-[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]propenamide

[0448] To a solution of the compound obtained in Step 7 (10 g, 27.8 mmol) in dichloromethane (50mL) are added N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (5.4 g, 28 mmol), 1- hydroxybenzotriazole hydrate (3.8 g, 28 mmol), N,N-diisopropylethylamine (6.9 mL, 41 mmol) and a solution of 2 M dimethylamine in THF (20.8 mL). The reaction mixture is stirred overnight at room temperature, diluted with dichloromethane, washed successively with water, aqueous 1M HCl solution and brine. The 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.

[0449] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.68 (d, 2H), 7.31 (d, 2H), 7.15-7 (m, 4H), 4.65 / 4.25(2d, 2H), 4.19 (m, 1H), 2.88 / 2.78 (2s, 6H), 2.68 / 2.59 (2dd, 2H), 2.33 (s, 3H), 2.25 (t, 2H), 1.5 (quad, 2 H).

[0450] Step 9: N,N-dimethyl-3-[(3R)-2-(p-tolylsulfonyl)-3,4-dihydro-1H-isoquinolin-3-yl]propan-1-amine

[0451] To a solution of Step 8 (6.6 g, 17.1 mmol) in anhydrous THF (70 mL) is added dropwiseunder 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 NaHCO3solution, brine, and dried over MgSO4and concentrated. The crude mixture is purified by chromatography over silica gel using dichloromethane and NH32M in ethanol as eluants, affording the desired compound.

[0452] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.66 (d, 2H), 7.32 (d, 2H), 7.16-7 (m, 4H), 4.63 / 4.21(dd, 2H), 4.15 (m, 1H), 2.67 / 2.55 (2dd, 2H), 2.33 (s, 3H), 2.1 (t, 2H), 2.04 (s, 6H), 1.35 / 1.27 (2m, 4H). 95 ME150999036v.1 132043-01120

[0453] Step 10: N,N-dimethyl-3-[(3R)-1,2,3,4-tetrahydroisoquinolin-3-yl]propan-1-amine

[0454] To a solution of naphthalene (13 g, 103 mmol) in anhydrous THF (35 mL) under argonatmosphere is added sodium (2.4 g, 103 mmol) portionwise. After stirring 1h at room temperature, 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. 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 NH32M in ethanol as eluants, affording the desired compound.

[0455] 1H NMR: ^ (400 MHz; dmso-d6; 300K): 7.17-7.04 (m, 4H), 4 (s, 2H), 2.92 (m, 1H),2.82 / 2.53 (2dd, 2H), 2.32 (m, 2H), 2.22 (s, 6H), 1.65-1.5 (m, H).

[0456] 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

[0457] To a solution of the compound obtained in Step 3 (15.5 g, 48 mmol) in dichloromethane (3L)are added N-(3-dimethylaminopropyl)-N′-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). The 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.

[0458] 1H NMR: ^ (500 MHz; dmso-d6; 300K): 7.6-7.25 (m, 3H), 7.2-6.85 (m, 4H), 6.55-6.15 (5s,1H), 5.35-3.75 (m, 2H), 4.85 / 4.75 / 3.6 / 3.55 (4m, 1H), 4.1 (m, 2H), 3.45 / 3.2 (2s, 3H), 3-1.8 (m, 2H), 2.5-2 (3s, 3H), 2.2-1.8 (m, 2H), 2.05-1.9 (4s, 6H), 1.4 / 1.1 / 0.9 (3m, 2H), 1.4 / 1.25 / 1.1 (m, 2H), 1.2 / 1.1 (2t, 3H).

[0459] MS (ESI): m / z 521 [M +H]+.

[0460] 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

[0461] 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. Dichloromethane and 1M HCl solution are added to the reaction mixture. The organic phase is separated, dried over MgSO4and concentrated to dryness. The residue is purified by chromatography over silica gel using dichloromethane and methanol as eluants, affording the desired compound.

[0462] MS (ESI): m / z 494 [M]+.96 ME150999036v.1 132043-01120

[0463] 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

[0464] 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 µL, 1.3 mmol). The reaction is stirred at room temperature overnight. To the reaction are added successively 4-[4- [tert-butyl(dimethyl)silyl]oxyanilino]-1,5-dimethyl-pyrrole-2-carbonitrile (500 mg, 1.5 mmol prepared using the procedure described in WO2015 / 011400, Preparation 18’’), pyridine (0.5 mL, 5.8 mmol) and 1,2-dichloroethane (60 mL). The reaction mixture is heated to reflux for 2h, cooled to room temperature, filtrated on Celite®, concentrated to dryness and purified by chromatography over silica gel using dichloromethane and NH32M methanol as eluants, affording the desired compound.

[0465] 1H NMR: ^ (500 MHz; dmso-d6; 300K): 7.55-6.9 (m, 7H), 6.9-6.6 (m, 4H), 6.8-6.5 (m, 1H),5.45-5.1 (m, 1H), 5.35-4 (m, 2H), 4.8-3.5 (m, 1H), 3.7-3.1 (m, 6H), 2.75-2.5 (m, 2H), 2.45-1.8 (m,6 H), 2.3-1.9 (m, 2H), 2.15-1.95 (m, 6H), 1.45-1.3 (m, 4H), 0.85 (s, 9H), 0.15 (s, 6H).

[0466] 13C NMR: ^ (500 MHz; dmso-d6; 300K): 130.9-124, 127.6, 119.7, 116.9, 110.5 / 109.5, 56.2,51.9 / 46.7 / 45.3, 44.1 / 40.1, 42, 33, 31.8, 31.5 / 31.1, 26.8 / 20.4, 25.6, 11.4, 9.5, -4.5.

[0467] HRMS (ESI): [M+H]+= 817.4020

[0468] 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

[0469] To a solution of the compound obtained in Step 13 (0.5 g, 0.61 mmol) in THF (20 mL) isadded 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.

[0470] 1H NMR: ^ (500 MHz; dmso-d6; 300K): 9.35 (broad s, 1H), 7.55-6.85 (m, 7H), 6.95 / 6.75(2d, 2H), 6.75-6.5 (m, 1H), 6.65-6.55 (m, 2H), 5.8-5.25 (m, 1H), 5.3-3.6 (m, 2H), 4.8 / 4.75 / 3.45 (3m, 1H), 3.65-3.5 (m, 3H), 3.45-3.05 (m, 3H), 2.65-1.65 (m, 2H), 2.4-1.95 (m, 3H), 2.25-1.75 (m, 2H), 2.15-1.75 (m, 3H), 2.1-1.95 (4s, 6H), 1.45-0.6 (m, 4H).

[0471] 13C NMR: ^ (500 MHz; dmso-d6; 300K): 130-126, 128, 117, 116, 111-110, 59, 53 / 47 / 46, 45-40, 45, 33, 32-31, 32, 29-24, 12, 10.

[0472] HRMS (ESI): [M+H]+=703.3155 (703.3158 calculated).

[0473] Preparation of P3: N-[1-(difluoromethyl)pyrazol-4-yl]-5-[2-[2-[4-(2-morpholinoethoxy)phenyl]acetyl]-7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-3,4-dihydro-1H- isoquinolin-6-yl]-N-(4-hydroxyphenyl)-1,2-dimethyl-pyrrole-3-carboxamide 97 ME150999036v.1 132043-01120

[0474] Step A: N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-1-(difluoromethyl)pyrazol-4-amine

[0475] Using General procedure 1b, starting from 4-bromo-1-(difluoromethyl)pyrazole (1.00 g, 5.08mmol) as the appropriate aryl-bromide and Preparation IIIa (1.19 g, 1.05 eq.) as the appropriate aniline, afforded the title compound (1.19 g, 69%).

[0476] 1H NMR (500 MHz, DMSO-d6) δ ppm 7.99 (s, 1H), 7.72 (s, 1H), 7.67 (t, 1H), 7.63 (s, 1H),6.79 (d, 2H), 6.70 (d, 2H), 0.93 (s, 9H), 0.14 (s, 6H);13C NMR (125 MHz, DMSO-d6) δ ppm 147.7, 139.7, 136.3, 129.6, 120.8, 115.5, 115.4, 111.3, 26.1, 18.4, -4.1.

[0477] HRMS-ESI (m / z) [M+H]+ calcd for C16H24F2N3OSi: 340.1651, found 340.1650.

[0478] Step B: 9H-fluoren-9-ylmethyl 6-[4-[[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-[1-(difluoromethyl)pyrazol-4-yl]carbamoyl]-1,5-dimethyl-pyrrol-2-yl]-7-[(3R)-3-methyl-3,4-dihydro- 1H-isoquinoline-2-carbonyl]-3,4-dihydro-1H-isoquinoline-2-carboxylate

[0479] Using the procedure described in Preparation VIIb Step C and the product from Step A (1.16g, 3.42 mmol) as the appropriate aniline instead of the product of Preparation IVa Step B afforded the title product (2.18 g, 77 %).

[0480] 1H NMR (500 MHz, DMSO-d6) δ ppm 8.3-6.53 (m, 21 H), 5.34-4.7 (s, 1 H), 5.33-2.07 (m,14 H), 3.39-3.03 (s, 3 H), 2.48-2.1 (s, 3 H), 1.12-0.48 (brd, 3 H), 0.86-0.75 (s, 9 H), 0.12-0 (s, 6 H).

[0481] HRMS-ESI (m / z) [M+H]+ calcd for C58H61F2N6O5Si: 987.4435, found 987.4438.

[0482] Step C: N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-N-[1-(difluoromethyl)pyrazol-4-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

[0483] Using the procedure described in Preparation VIIb Step D and the product from Step B (2.18g, 2.21 mmol) instead of the product from Preparation VIIb Step C afforded the title product (0.83 g, 49 %).

[0484] 1H NMR (500 MHz, DMSO-d6) δ ppm 8.24-8.02 (s, 1 H), 7.73 (t, 1 H), 7.54-7.37 (s, 1 H),7.26-6.55 (m, 10 H), 5.3-4.67 (s, 1 H), 5.26-2.09 (m, 12 H), 3.35-3.02 (s, 3 H), 2.46-2.08 (s, 3 H), 1.04-0.52 (d, 3 H), 0.86 (s, 9 H), 0.17-0.04 (s, 6 H).

[0485] HRMS-ESI (m / z) [M+H]+ calcd for C43H51F2N6O3Si: 765.3754, found 765.3756.98 ME150999036v.1 132043-01120

[0486] Step D: N-[4-[tert-butyl(dimethyl)silyl]oxyphenyl]-N-[1-(difluoromethyl)pyrazol-4-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

[0487] Using modified General procedure 4a (using TBTU instead of HATU and omittingpreparative HPLC flash chromatography), starting from the product of Preparation Va (283 mg, 1.2 eq.) as the appropriate carboxylic acid and the product from Step C (680 mg, 0.889 mmol) as the appropriate amine, afforded the title compound (643 mg, 71%).

[0488] 1H NMR (500 MHz, DMSO-d6) δ ppm 8.22-8.04 (s, 1 H), 7.72 (t, 1 H), 7.53-7.36 (s, 1 H),7.26-6.58 (m, 14 H), 5.29-2.08 (m, 13 H), 5.28-4.73 (s, 1 H), 4.05 (t, 2 H), 3.56 (m, 4 H), 3.35-3.01 (s, 3 H), 2.66 (t, 2 H), 2.47-2.09 (s, 3 H), 2.45 (m, 4 H), 1.04-0.49 (d, 3 H), 0.89-0.8 (s, 9 H), 0.13-0.01 (s, 6 H).

[0489] HRMS-ESI (m / z) [M+H]+ calcd for C57H68F2N7O6Si: 1012.4963, found 1012.4968.

[0490] Step E: N-[1-(difluoromethyl)pyrazol-4-yl]-5-[2-[2-[4-(2-morpholinoethoxy) phenyl]acetyl]-7-[(3R)-3-methyl-3,4-dihydro-1H-isoquinoline-2-carbonyl]-3,4-dihydro-1H-isoquinolin-6-yl]-N-(4- hydroxyphenyl)-1,2-dimethyl-pyrrole-3-carboxamide

[0491] To the product from Step D (100 mg, 0.099 mmol) dissolved in acetonitrile (150 mL / mmol)2M aq. NaOH solution (6 eq.) was added and the resulting mixture was stirred at rt. After reaching complete conversion the pH was adjusted to 7 by 2M aq. HCl solution and the product was partitioned between DCM and water. The phases were separated, and the organic phase was dried over MgSO4and concentrated in vacuo. The crude material was purified by automated flash chromatography using DCM and MeOH as eluents to afford the title compound (19.5 mg, 22%).

[0492] HRMS-ESI (m / z) [M+H]+ calcd for C51H54F2N7O6: 898.4104, found 898.4152.

[0493] Preparation of P4: 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 99 ME150999036v.1 132043-01120

[0494] 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

[0495] Using the procedure described in Step D of the Preparation of P3, starting from the product ofPreparation 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%).

[0496] HRMS-ESI (m / z) [M+H]+ calcd for C60H72N7O6Si: 1014.5308, found 1014.5312.

[0497] 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

[0498] 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. NaOH solution to afford the title compound (299 mg, 86%).

[0499] HRMS-ESI (m / z) [M+H]+ calcd for C54H51N7O6: 900.4443, found 900.4445.Example 2. Synthesis and Characterization of Linkers, Linker-Payload, and Precursors Thereof 100 ME150999036v.1 132043-01120

[0500] Linker-payloads P1-L12-P2, P1-L12-P3, P1-L12-P4, P1-L17-P2, P1-L17-P3, P1-L17-P4, andP1-L19-P2 were prepared according to the methods described in International PCT PCT / US2023 / 022990, which is incorporated herein by reference, and as described herein. Below is a table showing structures of these Linker-payloads. 101 ME150999036v.1 132043-01120

[0501] Materials, Methods & General Procedures:

[0502] All reagents obtained from commercial sources were used without further purification.Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed on CombiFlash Rf (Teledyne ISCO) with pre-packed silica-gel cartridges (Macherey-Nagel Chromabond Flash). Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60 F254 silica-gel. Microwave heating was performed in CEM Discover® instrument.

[0503] NMR data were acquired at a temperature of 298K on a Bruker Avance NMR spectrometerequipped with a 5 mm BBFO CryoProbe with z-gradient operating at a frequency of 400.13 MHz for1H, 376.50 MHz for19F, 100.61 MHz for13C. Chemical shifts for the1H and13C spectra were referenced by setting internal tetramethylsilane (TMS) to 0 ppm. Analytical Methods LC / MS data was acquired using an instrument with the following parameters: Pump Waters AcQuity UPLC Binary Solvent ManagerSample Manager Waters AcQuity UPLC Sample Manager Column Compartment Waters AcQuity UPLC Column Manager Detector Waters AcQuity UPLC PDA ELSD Shimadzu ELSD-LTIIMass Spec Waters SQD Columns AcQuity UPLC BEH C181.7µm 2.1x50mm Eluent A1 0.1% Formic Acid in Water Eluent B1 0.1% Formic Acid in AcetonitrileEluent A2 5mM Ammonium Hydroxide in WaterEluent B2 5mM Ammonium Hydroxide in Acetonitrile The methods used to generate LC / MS data were as follows: 2 min acidic method: 102 ME150999036v.1 132043-01120 2 min basic method: 5 min acidic method 103 ME150999036v.1 132043-01120 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 Detector Waters AcQuity UPLC PDA ELSD n / aMass Spec Waters Xevo G2 Qtof AcQuity UPLC PrST C4300Å 1.7 µm 2.1x100mm AcQuity UPLC CSH C181.7 µm 2.1x50mm ProSwift RP-3U 4.6x50mm SS Eluent A1 0.1% Formic Acid in Water Eluent B1 0.1% Formic Acid in AcetonitrileEluent 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 ManagerColumn Compartment Waters AcQuity UPLC Column ManagerDetector Waters AcQuity UPLC PDA ELSD n / a Mass Spec Waters Xevo G2 Qtof AcQuity UPLC PrST C4300Å 1.7µm 2.1x100mm Columns AcQuity UPLC CSH C181.7µm 2.1x50mm POROS R110µm 2.1 x 100mm Eluent A1 0.1% Formic Acid in Water Eluent B1 0.1% Formic Acid in 80:20Isopropanol:Acetonitrile Eluent A2 0.05% Triflouroacetic Acid in Water Eluent B2 0.05% Triflouroacetic Acid in Acetonitrile The method used to generate HRMS data for linker / payloads and synthetic intermediates was as follows: 104 ME150999036v.1 132043-01120 Peptide300-10000Da5min QT1 Peptide300-4000Da5min QT2 105 ME150999036v.1 132043-01120 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. Four Prep-HPLC methods were used: a. TFA method: solvent: A water + 0.05 % TFA, B acetonitrile + 0.05 % TFA, gradientfrom 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 to30 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. Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

[0504] 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. The solid was filtered and washed with water twice (600 mL) and dried under high vacuum to get a residue. The product 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, crude) was obtained as 106 ME150999036v.1 132043-01120 white solid. LCMS: RT = 0.537 min, MS m / z = 193.2.1H NMR: 400 MHz DMSO δ 8.57 (br d, J = 4.4 Hz, 1H), 8.31 (dd, J = 2.4, 8.6 Hz, 1H), 8.21 (d, J = 2.4 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 5.54 (t, J = 5.6 Hz, 1H), 4.72 (d, J = 5.5 Hz, 2H), 2.78 (d, J = 4.4 Hz, 3H). 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 BH3-Me2S (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. After completion, 4M HCl (1200 mL) in Methanol was added to the reaction mixture at 0 °C and heated at 65 °C for 8 h. The reaction mixture was cooled to 0 °C, the solid was filtered and concentrated in reduce pressure. (2-((methylamino)methyl)-4-nitrophenyl)methanol was obtained as a white solid (520 g). LCMS: RT = 0.742 min, MS m / z = 197.1 [M+H]+.1H NMR: 400 MHz DMSO δ 9.25 (br s, 2H), 8.37 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 2.4, 8.5 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 5.72 (br s, 1H), 4.65 (s, 2H), 4.15 (br s, 2H), 2.55 - 2.45 (m, 3H) Step 3: Synthesis of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N- methylmethanamine A solution of (2-((methylamino)methyl)-4-nitrophenyl)methanol (520 g, 2.65 mol) and imidazole (721 g, 10.6 mol) in DCM (2600 mL) was cooled to 0°C then TBDPS-Cl (1.09 kg, 3.98 mol, 1.02 L) was added drop wise and the mixture was stirred for 2 h. The mixture was poured into ice cold water (1000 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated under vacuum to give a crude product. The crude product was purified by chromatography on a silica gel eluted with ethyl acetate:Petroleum ether (from 10 / 1 to 1) to give a residue. 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N- methylmethanamine (600 g) was obtained as a yellow liquid. LCMS: product: RT = 0.910 min, MS m / z = 435.2 [M+H]+ 1H NMR: 400 MHz CDCl3 δ 8.23 (d, J=2.4 Hz, 1H), 8.15 (dd, J=2.4, 8.4 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.71 - 7.66 (m, 4H), 7.50 - 7.37 (m, 6H), 4.88 (s, 2H), 3.65 (s, 2H), 2.39 (s, 3H), 1.12 (s, 9H) Step 4: Synthesis of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5- nitrobenzyl)(methyl)carbamate 107 ME150999036v.1 132043-01120 To a solution of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N- methylmethanamine (400 g, 920.3 mmol) in THF (4000 mL) was added Fmoc-OSU (341.5 g, 1.01 mol) and Et3N (186.2 g, 1.84 mol, 256.2 mL), and the mixture was stirred at 25 °C for 1 h. The mixture was poured into water (1600 mL) and extracted twice with ethyl acetate (1000 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated under vacuum to give crude product. The crude product was purified by chromatography on a silica gel eluted with petroleum ether:ethyl acetate (from 1 / 0 to 1 / 1) to give (9H-fluoren-9-yl)methyl (2-(((tert- butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (405 g) as a white solid. LCMS: RT = 0.931 min, MS m / z = 657.2 [M+H]+. 1H NMR: 400 MHz CDCl3 δ 8.21 - 7.96 (m, 1H), 7.87 - 7.68 (m, 3H), 7.68 - 7.62 (m, 4H), 7.62 - 7.47 (m, 2H), 7.47 - 7.28 (m, 9H), 7.26 - 7.05 (m, 2H), 4.81 (br s, 1H), 4.62 - 4.37 (m, 4H), 4.31 - 4.19 (m, 1H), 4.08 - 3.95 (m, 1H), 2.87 (br d, J = 5.2 Hz, 3H), 1.12 (s, 9H). Step 5: Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate A solution of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5- nitrobenzyl)(methyl)carbamate (3.0 g, 4.57 mmol, 1.0 equiv.) in MeOH (90 mL) and EtOAc (30 mL) was degassed and purged to a balloon of N2via three way stopcock. After repeating degas / N2purge 2x, 10% Pd / C deGussa type (0.486 g, 0.457 mmol, 0.1 equiv.) was added. The resulting mixture was degassed and purged to a balloon of 2 H2via three way stopcock. After repeating degas / H2purge 2x, the reaction stirred under the balloon pressure of H2for 4 hours. The reaction was degassed and purged to N2, filtered through a pad of celite eluting further with MeOH. After removal of the volatiles in vacuo and pumping on high vac (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate was obtained (2.78 g, 97%). LCMS: MH+=627.7; Rt=1.59 min (2 min acidic method-Method A).1H NMR: 400 MHz CDCl3 δ 7.80 (br d, J = 7.2 Hz, 1H), 7.74 - 7.67 (m, 5H), 7.64 (br d, J = 6.8 Hz, 1H), 7.49 - 7.30 (m, 10H), 7.23 - 7.06 (m, 2H), 6.61 - 6.41 (m, 2H), 4.66 (br d, J = 7.2 Hz, 2H), 4.55 (s, 2H), 4.51 - 4.34 (m, 2H), 4.32 - 4.10 (m, 1H), 3.66 (br s, 2H), 2.96 - 2.78 (m, 3H), , 1.07 (s, 9H). Synthesis of (9H-fluoren-9-yl)methyl (S)-(5-(2-((tert-butoxycarbonyl)amino)-5- ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate 108 ME150999036v.1 132043-01120

[0505] A solution of (S)-2-((tert-Butoxycarbonyl)amino)-5-ureidopentanoic acid (17.57 grams, 63.8mmol), (9H-fluoren-9-yl)methyl (5-amino-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (40 grams, 63.8 mmol) and HOAT (1.0 M in DMA, 63.8 mL, 63.8 mmol) in DMF (36 mL) was stirred until homogeneous, cooled in a 0°C bath and EDC (12.23 grams, 63.8 mmol) was added. The reaction was left stirring as the bath was allowed to warm to rt overnight. After 16 hours, the reaction solution was dripped into 4L H2O with stirring over ca.30 minutes at which time the ppt was filtered, rinsed with H2O (1L) and air dried under vacuum. The wet cake was dissolved in 15% iPrOH / EtOAc (ca. 1.8 L) and washed with H2O (250 mL), with NaHCO3(sat.) (250 mL) and with NaCl(sat.) (250 mL), dried over MgSO4, filtered, concentrated, and pumped on to yield (9H-fluoren-9-yl)methyl (S)-(5-(2-((tert- butoxycarbonyl)amino)-5-ureidopentanamido)-2-(((tert- butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (54.47 grams, 96% yield). LC / MS: MH+=884.7, Rt=3.82 min (5 min. acidic method). Synthesis of (9H-fluoren-9-yl)methyl (S)-(5-(2-((tert-butoxycarbonyl)amino)-5- ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0506] To a solution of (9H-fluoren-9-yl)methyl (S)-(5-(2-((tert-butoxycarbonyl)amino)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (33.29 grams, 37.7 mmol) in THF (45 mL) was added AcOH (12.93 mL, 226 mmol) followed by 1.0 M TBAF in THF (56.6 mL, 56.6 mmol). After stirring for 72 hours, the volatiles were removed in vacuo and the residue was partitioned between 15% iPrOH / EtOAc (1.5 L) and H2O (300 mL), mixed, separated, washed further with H2O (5x350 mL), with NaHCO3(sat.) (2x300 mL), with NaCl(sat.) (300 mL), dried over MgSO4, filtered, concentrated, pumped on, triturated with Et2O and filtered to yield (9H- fluoren-9-yl)methyl (S)-(5-(2-((tert-butoxycarbonyl)amino)-5-ureidopentanamido)-2- (hydroxymethyl)benzyl)(methyl)carbamate (19.17 grams, 79% yield). LC / MS: MH+=646.7, Rf=2.28 min. (5 min acidic method). 109 ME150999036v.1 132043-01120 Synthesis of (9H-fluoren-9-yl)methyl (S)-(5-(2-amino-5-ureidopentanamido)-2- carbamate

[0507] (9H-fluoren-9-yl)methyl (S)-(5-(2-((tert-butoxycarbonyl)amino)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (15 g, 23.23 mmol) was dissolved in DCM (30 mL),cooled in ice bath and TFA (28.5 mL, 372 mmol) was added slowly over 5 min. The reaction was kept stirring in the ice bath for 1 hr. The reaction was slowly warmed up to RT and stirred at RT for 1 hour. The volatiles were removed in-vacuo. The product was retaken in dicholoroethane (210 mL). The resulting solution was removed in-vacuo. The process was repeated 2 more times (3 times in total) to obtain a dark yellow solid. The solid was suspended in diethyl ether (300 mL). It was sonicated for 10 min then decanted. The process was repeated 2 more times. The solid was collected by vacuum filtration. After air drying overnight under vacuum, the solid was dissolved in MeOH (100 mL) and this solution was added dropwise over ca. 20 minutes to 7N ammonia in MeOH (201 ml, 402 mmol). After stirring for 30 minutes, the volatiles were removed in-vacuo and, after trituration with Et2O, (9H-fluoren-9-yl)methyl (S)-(5-(2-amino-5-ureidopentanamido)-2- (hydroxymethyl)benzyl)(methyl)carbamate (15.75 grams) was obtained. LC / MS: MH+=546.5, Rt=1.43 min. (5 min acidic method). The material was used as is for next step. Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0508] Boc-Val-OH (6.47 g, 29.8 mmol), EDC.HCl (5.48 g, 28.7 mmol) and HOAT (3.89 g, 28.7mmol) were dissolved in DMF (35 mL) and stirred at RT for 10 min. at which time (9H-fluoren-9- yl)methyl (S)-(5-(2-amino-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (13 g, 23.83 mmol) was added. DIPEA (4.15 mL, 23.83 mmol) was added. After stirring for 30 minutes the reaction mixture was added dropwise to 4L of water under vigorous stirring. After 30 minutes, the precipitate was collected by vacuum filtration. The cake was washed with water (1200 mL). The product was air dried under vacuum, was triturated with MTBE, and dried under vacuum for to yield (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (12.8 g, 72% yield). LC / MS: MH+=745.7, 2.37 min. (5 min acidic method). 110 ME150999036v.1 132043-01120 Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate

[0509] To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (1.0 gram, 1.342 mmol) in THF(20 mL) was added NaHCO3(677 mg, 8.05 mmol)(6eq), then cooled to 0oC in ice-water bath, followed by adding thionyl chloride (0.245 mL, 3.36 mmol) (2.5eq) slowly. Themixture was stirred at 0oC for 15 min, then at RT for 1h. The reaction was paritioned between EtOAcand NaHCO3 (sat.), separated, washed with NaCl (sat.), dried over MgSO4 and the volatiles were removed in vacuo. The residue was purified by ISCO SiO2chromatography (0-30% iPrOH / CH2Cl2) to yield (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+=763.2; Rt=1.18 min (2 min acidic method). Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

[0510] To a solution of (9H-fluoren-9-yl)methyl (S)-(5-(2-amino-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (9.4 g, 14.25 mmol), HOAT (2.32 g, 17.1 mmol) and ((allyloxy)carbonyl)-L-valine (3.44 g, 17.10 mmol) in DMF (27 mL) was added DIEA (5.53 g, 42.7 mmol) and EDC (3.28 g, 17.1 mmol). After stirring for 2 hours, the solution was dripped into 3L of H2O with stirring. The solid formed was collected by vacuum filtration, rinsed further with H2O and air dried under vacuum to yield (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2- (hydroxymethyl)benzyl)(methyl)carbamate (7.69 g, 74% yield). LC / MS: MH+=729.6, Rt=2.27 min (5 min acidic method). Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate 111 ME150999036v.1 132043-01120

[0511] To a heterogeneous solution of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2- (hydroxymethyl)benzyl)(methyl)carbamate (4.0 g, 5.5 mmol) and NaHCO3(2.8 g, 33 mmol) in THF (55 mL) at rt was added thionyl chloride (1.0 mL, 14 mmol). After stirring for 1 hour, the solution was partitioned between EtOAc and NaHCO3(sat.), extracted further with EtOAc, dried over MgSO4, filtered, concentrated, purified by SiO2 chromatography (0-20% iPrOH / CH2Cl2 eluant). After concentration, (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (2.1 g, 51% yield) was obtained. LC / MS: MH+=747.6, Rt=2.72 min (5 min acidic method). Synthesis of N-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-3-((R)-2-(2-(4-((4-((tert- butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5-dimethyl- 1H-pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1- aminium trifluoroacetate

[0512] A solution of (R)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-(5-chloro-2-(3-(3-(dimethylamino)propyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N-(5-cyano-1,2-dimethyl- 1H-pyrrol-3-yl)-1,2-dimethyl-1H-pyrrole-3-carboxamide (400 mg, 0.489 mmol), tert-butyl ((S)-1- (((S)-1-((4-(chloromethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.497 g, 0.685 mmol) and DIEA (426 µL, 2.45 mmol) in DMSO (2 mL) was stirred at rt for 16 hours. The solution was purified by ISCO C18 RP- HPLC. Upon lyophilization, N-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)- 5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-3-((R)-2-(2-(4-((4-((tert- butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5-dimethyl-1H- pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium 112 ME150999036v.1 132043-01120 trifluoroacetate (575 mg, 87%) was obtained. HRMS: M+=1346.4700, Rt=3.22 min (5 min acidic method). Synthesis of N-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2- ((prop-2-yn-1-yloxy)methyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3- yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4- tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate

[0513] To a solution of N-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-3-((R)-2-(2-(4-((4-((tert- butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5-dimethyl-1H- pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate ( 930 mg, 690 µmol) in MeOH (8 mL) was added HCl(conc.) (1.13 mL, 13.81 mmol). After stirring for 17 hours, the volatiles were removed in vacuo. The residue was dissolved in DMSO, purified by RP-HPLC and after lyophilization, N-(4-((S)-2-((S)-2-amino-3- methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-3-((R)-2-(4-chloro- 2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol- 2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (584 mg, 75% yield) was obtained. HRMS: M+=1132.4000, Rt=1.90 min (5 min acidic method). Synthesis of N-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)- 2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-3- ((R)-2-(2-(4-((4-((tert-butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3- yl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3- yl)-N,N-dimethylpropan-1-aminium trifluoroacetate 113 ME150999036v.1 132043-01120

[0514] A solution of (R)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-5-(5-chloro-2-(3-(3-(dimethylamino)propyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N-(5-cyano-1,2-dimethyl- 1H-pyrrol-3-yl)-1,2-dimethyl-1H-pyrrole-3-carboxamide (1.0 g, 1.224 mmol), (9H-fluoren-9- yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (1.214 g, 1.590 mmol), tetrabutylammonium iodide (496 mg, 1.346 mmol) and DIEA (474 mg, 3.67 mmol) in DMSO (6 mL) was stirred at rt for 2 hours. The solution was purified by ISCO C18 RP-HPLC. Upon lyophilization, N-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2- ((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-3-((R)-2-(2-(4-((4- ((tert-butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5-dimethyl- 1H-pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1- aminium trifluoroacetate (1.414 g, 74%) was obtained. HRMS: M+=1543.8101, Rt=2.64 min (5 min acidic method). Synthesis of N-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)- 2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-3-((R)-2-(4-chloro-2-(4-((5- cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2- yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate

[0515] To a solution of N-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-3-((R)-2- (2-(4-((4-((tert-butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5- 114 ME150999036v.1 132043-01120 dimethyl-1H-pyrrol-2-yl)-4-chlorobenzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan- 1-aminium trifluoroacetate (952 mg, 0.574 mmol) in EtOH (4 mL) was added 4 M HCl in dioxane (0.94 mL, 11.5 mmol). After stirring for 3 hours, the volatiles were removed in vacuo, the residue was dissolved in DMSO and was purified by ISCO C18 RP-HPLC. Upon lyophilization, N-(2- (((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3- methylbutanamido)-5-ureidopentanamido)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl- 1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4- tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (664 mg, 81%) was obtained. HRMS: M+=1329.6700, Rt=2.07 min (5 min acidic method). Synthesis of N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-((methylamino)methyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2- dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)- 1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate

[0516] To a solution of N-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-3-((R)-2-(4-chloro-2-(4-((5- cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2- yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (316 mg, 0.22 mmol) and N-allylcarbonyloxy succinimide (87 mg, 0.437) in DMF (1 mL) was added DIEA (141 mg, 1.09 mmol). After stirring for 2 hours, 2.0 M Dimethyl amine in MeOH (1.09 mL, 2.18 mmoL) was added. After stirring for 45 minutes, the solution was diluted with DMSO and purified by ISCO RP-HPLC. Upon lyophilization, N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-3-((R)-2-(4-chloro-2-(4- ((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2- yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (208 mg, 79% yield ) was obtained. HRMS: M+=1191.6100, Rt=1.83 min (5 min acidic method). Synthesis of N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- 115 ME150999036v.1 132043-01120 hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin- 3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate

[0517] GENERAL PROCEDURE #1: to a solution of bis(4-nitrophenyl) carbonate (50.6 mg, 166µmol), tert-butyl 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72- tetracosaoxapentaheptacontan-75-oate (200 mg, 166 µmol) in DMF (2 mL) was added DIEA (58 µL, 333 µmol). After stirring for 1 hour, N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-3-((R)-2-(4-chloro-2-(4- ((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2- yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (207 mg, 158 µmol) was added to the solution as well as additional DIEA (58 µL, 333 µmol). After stirring for an additional 2 hours, the solution was diluted with DMSO and purified by ISCO RP- HPLC. Upon lyophilization, N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3- yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (335 mg, 84 % yield) was obtained. HRMS: (M+-H++Na+)+=2441.2800, Rt=2.46 min (5 min acidic method). Synthesis of N-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2- (2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin- 3-yl)-N,N-dimethylpropan-1-aminium trifluoroacetate 116 ME150999036v.1 132043-01120

[0518] GENERAL PROCEDURE #2: to a solution of N-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,81,81-trimethyl-3,79- dioxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3- yl)-N,N-dimethylpropan-1-aminium trifluoroacetate (214 mg, 84 µmol) in THF (6 mL) was added N,N,1,1,1-pentamethylsilanamine (59 mg, 505 µmol). An N2 balloon with 3-way stopcock was attached and house vacuum was pulled and reaction purged to N2 (repeated 2 x). Tetrakis(triphenylphosphine)palladium (19.5 mg, 17 µmol) was added, followed by degassing / purging to N2 as above (3x). After stirring for one hour, the volatiles were removed in vacuo, the residue was dissolved in DMSO and purified by C18 RP-ISCO. After lyophilization, N- (4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-3-((R)-2-(4-chloro-2-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)benzoyl)-1,2,3,4-tetrahydroisoquinolin-3- yl)-N,N-dimethylpropan-1-aminium trifluoroacetate was obtained (134 mg, 62%). HRMS: M+=2335.3000, Rt=2.17 min (5 min acidic method). Variations of this general method included reducing the equivalents of tetrakis(triphenylphosphine)palladium used, use of alternative amine nucleophiles (pyrrolidine, morpholine) as well as running the reaction in 1:1 CH2Cl2 / MeOH with the omission of an amine nucleophile specifically in cases when an FMOC was present. Synthesis of 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate 117 ME150999036v.1 132043-01120

[0519] Following GENERAL PROCEDURE #1 using bis(4-nitrophenyl) carbonate (92.6 mg,303.4 µmol), tert-butyl 1-amino- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan- 75-oate (366 mg, 304.4 µmol), DIEA (110 µL, 634 µmol) and then 4-(4-((S)-2-((S)-2- (((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,81,81-trimethyl-3,79- dioxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium Trifluoroacetate (513 mg, 317 µmol) and DIEA (110 µL, 634 µmol), 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium Trifluoroacetate (688 mg, 79%) was obtained. HRMS: M+=2616.4500, Rt=2.42 min (5 min acidic method). Synthesis of 4-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2- (2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate 118 ME150999036v.1 132043-01120

[0520] Following GENERAL PROCEDURE #2 using 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,81,81-trimethyl-3,79- dioxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate (688 mg, 251.8 µmol), N,N,1,1,1- pentamethylsilanamine (242 µL, 1511 µmol) and tetrakis(triphenylphosphine)palladium (14.5 mg, 12.7 µmol), 4-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(2,81,81- trimethyl-3,79-dioxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80- pentacosaoxa-2,4-diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3- yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate was obtained (486 mg, 69%). HRMS: M+=2532.4099, Rt=2.15 min (5 min acidic method). Synthesis of 4-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)- 2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4- (2-(4-(2-(6-(4-((4-((tert-butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3- yl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4-tetrahydroisoquinoline-2- carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate 119 ME150999036v.1 132043-01120

[0521] A solution of (R)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-1,2-dimethyl-5-(7-(3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-(2-(4-(2- morpholinoethoxy)phenyl)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)-1H-pyrrole-3-carboxamide (200 mg, 197 µmol), (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (196 mg, 257 µmol) and sodium iodide (35.5 mg, 237 µmol) in DMSO (1 mL) was heated at 30°C for 36 hours. Upon cooling, the solution was purified by ISCO C18 RP-HPLC. After lyophilization, 4-(2-(((((9H- fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)- 3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(2-(6-(4-((4-((tert- butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5-dimethyl-1H- pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin- 2(1H)-yl)-2-oxoethyl)phenoxy)ethyl)morpholin-4-ium Trifluoroacetate (249 mg, 67%) was obtained. HRMS: M+=1740.9100, Rt=3.26 min (5 min acidic method). Synthesis of 4-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)- 2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano- 1,2-dimethyl-1H-pyrrol-3-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)- 3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate

[0522] A solution of 4-(2-(((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(2- (6-(4-((4-((tert-butyldimethylsilyl)oxy)phenyl)(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)carbamoyl)-1,5- dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4- dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate (308 mg, 165.9 µmol) in MeOH (3 mL) was cooled in ice bath and 4M HCl in dioxane (3 mL, 12 mmol) was added. After stirring for one hour, the volatiles were removed in vacuo. The residue was dissolved in DMSO and purified by ISCO C18 RP-HPLC. After lyophilization, 4-(2-(((((9H-fluoren-9- yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl)-4-(2-(4-(2-(6-(4-((5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)(4- 120 ME150999036v.1 132043-01120 hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium Trifluoroacetate was obtained (170 mg, 58%). HRMS: M+=1526.8101, Rt=2.02 min (5 min acidic method). Synthesis of 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(4-(2-(6-(4-((1-(difluoromethyl)-1H- pyrazol-4-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl- 1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate

[0523] A solution of (R)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-N-(1-(difluoromethyl)-1H-pyrazol-4-yl)-1,2-dimethyl-5-(7-(3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-2-(2-(4-(2- morpholinoethoxy)phenyl)acetyl)-1,2,3,4-tetrahydroisoquinolin-6-yl)-1H-pyrrole-3-carboxamide (600 mg, 593 µmol), (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (930 mg, 1245 µmol), tetrabutylammonium iodide (219 mg, 593 µmol) and DIEA (108 µL, 622 µmol) in DMF (2.1 mL) was stirred at rt for 60 hours. The solution was purified by ISCO C18 RP-HPLC. After lyophilization, the residue (630 mg) was dissolved in DMF (2 mL) and 2M dimethyl amine in MeOH (2 mL, ca. 4000 µmol) was added. After standing two hours, 1.0 M tetrabutyl ammonium fluoride (0.20 mL, 200 µmol) was added. After 60 minutes, the solution was diluted in DMSO and purified by C18 ISCO RP-HPLC. Upon lyophilization, 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(4-(2-(6-(4-((1- (difluoromethyl)-1H-pyrazol-4-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7- ((R)-3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate (518 mg, 52%) was obtained. HRMS: M+=1386.6899, Rt=1.83 min (5 min acidic method). Synthesis of 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)-2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- 121 ME150999036v.1 132043-01120 diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((1-(difluoromethyl)-1H-pyrazol-4-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate

[0524] Following GENERAL PROCEDURE #1 using bis(4-nitrophenyl) carbonate (93.1 mg, 306µmol), DIEA (111 µL, 637.5 µmol), ), tert-butyl 1-amino- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontan- 75-oate (368 mg, 306 µmol) and then 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(4-(2-(6-(4-((1- (difluoromethyl)-1H-pyrazol-4-yl)(4-hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7- ((R)-3-methyl-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium (515 mg, 319 µmol) and additional DIEA (111 µL, 637.5 mmol), 4-(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)- 2-(2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((1-(difluoromethyl)-1H-pyrazol-4-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate (696 mg, 79%) was obtained. HRMS: M+=2614.4099, Rt=2.47 min (5 min acidic method Synthesis of 4-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2- (2,81,81-trimethyl-3,79-dioxo- 7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,80-pentacosaoxa-2,4- diazadooctacontyl)benzyl)-4-(2-(4-(2-(6-(4-((1-(difluoromethyl)-1H-pyrazol-4-yl)(4- hydroxyphenyl)carbamoyl)-1,5-dimethyl-1H-pyrrol-2-yl)-7-((R)-3-methyl-1,2,3,4- tetrahydroisoquinoline-2-carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2- oxoethyl)phenoxy)ethyl)morpholin-4-ium trifluoroacetate 122 ME150999036v.1 1320...

Claims

132043-01120 CLAIMS 1. An antibody-drug conjugate of Formula (I),ME150999036v.1132043-01120whereinindicates the point of attachment to Ab; wherein Ab is an anti-CD74 antibody or an antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) and three light chain CDRs selected from the group consisting of ME150999036v.1132043-01120 1) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:12; 2) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:4, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; 3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:6, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:7, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:8; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; and 4) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:9, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; and wherein a is an integer from 1 to 16.

2. The antibody-drug conjugate of claim 1, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

18.

3. The antibody-drug conjugate of claim 1 or 2, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises: 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:20 or a sequence that is at least 95% identical to SEQ ID NO:

20.

4. The antibody-drug conjugate of any one of claims 1-3, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises one or more Fc silencing mutations. ME150999036v.1132043-01120 5. The antibody-drug conjugate of any one of claims 1-4, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises one or more CysMab mutations.

6. The antibody-drug conjugate of any one of claims 1-5, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises one or more CysMab mutations selected from E152C, S375C, or both E152C and S375C of the heavy chain of the antibody or antigen binding fragment thereof, wherein the position is numbered according to the EU system.

7. The antibody-drug conjugate of any one of claims 1-6, wherein the anti-CD74 antibody or antigen-binding fragment thereof comprises a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a single chain Fv (scFv), a disulfide-linked Fv (sdFv), a Fd fragment, a Fv fragment, a dAb fragment, a maxibody, a minibody, an intrabody, a diabody, a half antibody, or a one-arm antibody.

8. The antibody-drug conjugate of any one of claims 1-7, wherein the anti-CD74 antibody or antigen-binding fragment thereof is a monoclonal antibody.

9. The antibody-drug conjugate of any one of claims 1-8, wherein is10. The antibody-drug conjugate of any one of claims 1-8, wherein is: ME150999036v.1132043-01120.

11. The antibody-drug conjugate of any one of claims 1-8, wherein is.

12. The antibody-drug conjugate of any one of claims 1-8, wherein is .ME150999036v.1132043-0112013. The antibody-drug conjugate of any one of claims 1-8, wherein is.

14. The antibody-drug conjugate of any one of claims 1-8, wherein is15. The antibody-drug conjugate of any one of claims 1-8, wherein is ME150999036v.1132043-01120.

16. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 1 to 15, wherein the average a of the antibody-drug conjugates in the composition is from about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 2, or about 2 to about 4.

17. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1- 15, and a pharmaceutically acceptable carrier.

18. A method of treating a subject having or suspected of having a cancer, comprising administeringto the subject a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 15, the composition of claim 16, or the pharmaceutical composition of claim 17.

19. The method of claim 18, wherein the cancer expresses CD74.

20. The method of claim 18 or 19, wherein the cancer is a tumor or a hematological cancer.

21. The method of any one of claims 18-20, wherein 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. ME150999036v.1132043-01120 22. The method of any one of claims 18-21, wherein the cancer is acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

23. A method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-15 , the composition of claim 16, or the pharmaceutical composition of claim 17.

24. The method of claim 23, wherein the tumor expresses CD74.

25. The method of claim 23 or 24, wherein 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, spleen cancer, pancreatic cancer, stomach cancer, colon cancer, or head and neck cancer.

26. A method of reducing or inhibiting a hematological cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 15, the composition of claim 16, or the pharmaceutical composition of claim 17.

27. The method of claim 26, wherein the hematological cancer expresses CD74.

28. The method of claim 26 or 27, wherein 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).

29. The method of any one of claims 23 to 28, wherein administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor or 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%. ME150999036v.1132043-01120 30. 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 the antibody-drug conjugate of any one of claims 1 to 15, the composition of claim 16, or the pharmaceutical composition of claim 17.

31. The method of claim 30, wherein the cancer cell population expresses a CD74.

32. The method of claim 30 or 31, wherein the cancer cell population is from a tumor or a hematological cancer.

33. The method of any one of claims 30-32, wherein 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 head and neck cancer.

34. The method of any one of claims 30-33, wherein the cancer cell population is from acute myeloid leukemia, multiple myeloma, or B-cell lymphoma.

35. The method of any one of claims 30 to 34, wherein administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population or 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%.

36. The method of any one of claims 18 to 35, wherein the antibody-drug conjugate is administered as monotherapy.

37. The method of any one of claims 18 to 35, further comprising administering to the subject in needthereof at least one additional therapeutic agent and / or radiation therapy. ME150999036v.1132043-0112038. The method of claim 37, wherein the antibody-drug conjugate is administered adjunctive to the atleast one additional therapeutic agent and / or radiation therapy.

39. The method of claim 37 or 38, wherein the antibody-drug conjugate is administered in an amounteffective to sensitize the tumor cells to the at least one additional therapeutic agent and / or radiation therapy.

40. The method of claim 37, wherein the at least one additional therapeutic agent is a taxane, a vinca alkaloid, a MEK inhibitor, an ERK inhibitor, topoisomerase inhibitor, and / or a RAF inhibitor.

41. Use of an antibody-drug conjugate of any one of claims 1-15, a composition of claim 16, or a pharmaceutical composition of claim 17, for the manufacture of a medicament for (i) treating a subject having or suspected of having a cancer, (ii) reducing or inhibiting the growth of a tumor in a subject, (iii) reducing or inhibiting a hematological cancer in a subject, and / or (iv) reducing or slowing the expansion of a cancer cell population in a subject.

42. An antibody-drug conjugate of any one of claims 1-15, a composition of claim 16, or a pharmaceutical composition of claim 17 for use in (i) treating a subject having or suspected of having a cancer, (ii) reducing or inhibiting the growth of a tumor in a subject, (iii) reducing or inhibiting a hematological cancer in a subject, and / or (iv) reducing or slowing the expansion of a cancer cell population in a subject.

43. A process for producing an antibody drug conjugate comprising conjugating a linker-drug moiety to Ab to form an antibody-drug conjugate, wherein the linker-drug moiety is selected from the group consisting of: ME150999036v.1132043-01120ME150999036v.1132043-01120Ab is an anti-CD74 antibody or an antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) and three light chain CDRs selected from the group consisting of 1) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:12; 2) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:4, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID ME150999036v.1132043-01120 NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14 and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; 3) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:6, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:7, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:8; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; or 4) a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:9, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:5, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO:13, a light chain CDR2 (LCDR2) consisting of SEQ ID NO:14, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO:15; and a is an integer from 1 to 16. ME150999036v.1

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