WO2018213430A1

WO2018213430A1 - Anti-cd33 immunoconjugate dosing regimens

Info

Publication number: WO2018213430A1
Application number: PCT/US2018/032945
Authority: WO
Inventors: Russell Walker; Callum SLOSS; Patrick Zweidler-Mckay; Kerry CULM-MERDEK
Original assignee: Immunogen, Inc.
Priority date: 2017-05-17
Filing date: 2018-05-16
Publication date: 2018-11-22
Also published as: JP2020519640A; MA49152A; EP3624839A1; EP3624839A4

Abstract

The present invention provides a method of treating a cancer in a subject comprising administering to the subject an anti-CD33-immunoconjugate (e.g., IMGN779) at a therapeutically effective dose.

Description

ANTI-CD33 IMMUNOCONJUGATE DOSING REGIMENS

BACKGROUND OF THE INVENTION

[0001] Treatment of hematologic malignancies with conventional chemotherapies is often associated with significant morbidity because the chemotherapeutics are not specific for the cancerous cells. Furthermore, despite high initial response rates to chemotherapy, many patients with hematologic malignancies (e.g., acute myeloid leukemia) fail to achieve complete remission and relapse in a relatively short time period after diagnosis.

[0002] The leukocyte differentiation antigen CD33 is a 364 amino acid transmembrane glycoprotein with sequence homology to members of the sialoadhesin family, including myelin-associated glycoprotein and CD22, as well as sialoadhesin itself (S. Peiper, 2002, Leucocyte Typing VII, White Cell Differentiation, Antigens, Proceedings of the Seventh International Workshop and Conference, Oxford University Press, p. 777).

[0003] Expression of CD33 appears to be highly specific to the hematopoietic

compartment, with strong expression by myeloid precursor cells (S. Peiper, 2002). It is expressed by myeloid progenitor cells such as CFU-GEMM, CFU-GM, CFU-G and BFU-E, monocytes/macrophages, granulocyte precursors such as promyelocytes and myelocytes although with decreased expression upon maturation and differentiation, and mature granulocytes though with a low level of expression (S. Peiper, 2002). Anti-CD33 monoclonal antibodies have shown that CD33 is expressed by clonogenic, acute myelogenous leukemia (AML) cells in greater than 80% of human cases (LaRussa, V. F. et al, 1992, Exp. Hematol. 20:442-448). In contrast, pluripotent hematopoietic stem cells that give rise to "blast colonies" in vitro (Leary, A. G. et al., 1987, Blood 69:953) and that induce hematopoietic long-term marrow cultures (Andrews R. G. et al.., 1989, J. Exp. Med. 169: 1721; Sutherland, H. J. et al, 1989, Blood 74: 1563) appear to lack expression of CD33.

[0004] Due to the selective expression of CD33, immunoconjugates (also known as

antibody drug conjugates or ADCs) that combine cytotoxic drugs with monoclonal antibodies that specifically recognize and bind CD33 have been proposed for use in selective targeting of hematological malignancies (e.g., AML cells). However, after 10 years on the market, the anti-CD33 immunoconjugate Mylotarg® (Gemtuzumab ozogamicin) was voluntarily withdrawn from the market as a result of concerns related to safety and efficacy. Therefore, there is still a need to identify particular dosage regimens for particular anti-CD33 immunoconjugates for treating patients suffering from cancer, in particular hematologic cancers, such as, AML.

SUMMARY OF THE INVENTION

[0005] The present invention relates at least in part to the discovery that particular dosing regimens of anti-CD33 immunoconjugates (e.g., IMGN779) are useful in treating patients suffering from cancer, in particular hematologic cancers, such as, AML. Accordingly, methods of treating cancer using anti-CD33 immunoconjugates (e.g., IMGN779) are provided herein.

[0006] In one embodiment, a method of treating cancer in a subject comprises

administering to the subject about 1 to about 2 mg/kg of an anti-CD33 immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H;r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively and a variable light chain comprising the CDR1, CDR2, and CDR3 sequences set for the in SEQ ID NOs: 4-6, respectively.

[0007] In one embodiment, a method of treating cancer in a subject comprises

administering to the subject about 0.70 to about 1.25 mg/kg of an anti-CD33

immunoconjugate of the following formula:

[0008] In one embodiment, a method of treating cancer in a subject comprises

administering to the subject about 0.54 to about 1.0 mg kg of an anti-CD33

immunoconjugate of the following formula:

[0009] In one embodiment, a method of treating cancer in a subject comprises

administering to the subject about 0.54 to about 0.8 mg/kg of an anti-CD33

immunoconjugate of the following formula:

[0010] In one embodiment, about 0.54 to about 0.70 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.54 or about 0.70 mg/kg of the

immunoconjugate is administered.

[0011] In one embodiment, about 0.5 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.6 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.7 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.75 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.8 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.85 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.9 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.91 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.92 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.93 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.94 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.95 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.96 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.97 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.98 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.99 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.0 mg/kg of the immunoconjugate is administered.

[0012] In one embodiment, about 1.01 mg/kg of the immunoconjugate is administered.

In one embodiment, about 1.02 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.03 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.04 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.05 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.06 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.07 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.08 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.09 mg/kg of the immunoconjugate is administered.

[0013] In one embodiment, about 1.1 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.11 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.12 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.13 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.14 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.15 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.16 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.17 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.18 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.19 mg/kg of the immunoconjugate is administered.

[0014] In one embodiment, about 1.2 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.21 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.22 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.23 mg/kg of the immunoconjugate is administered.. In one embodiment, about 1.24 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.25 mg/kg of the immunoconjugate is administered. [0015] In one embodiment, about 1.5 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.51 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.52 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.53 mg/kg of the immunoconjugate is administered.. In one embodiment, about 1.54 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.55 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.56 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.57 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.58 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.59 mg/kg of the immunoconjugate is administered. In one embodiment, about 1.6 mg/kg of the immunoconjugate is administered.

[0016] In one embodiment, about 2 mg/kg of the immunoconjugate is administered.

[0017] In one embodiment, the immunoconjugate is administered about once in two weeks.

[0018] In one embodiment, a method of treating cancer in a subject comprises

administering to the subject about 0.30 to about 0.54 mg/kg of an anti-CD33

immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H; r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1 -3, respectively and a variable light chain comprising the CDRl, CDR2, and CDR3 sequences set for the in SEQ ID NOs: 4-6, respectively. [0019] In one embodiment, about 0.39 to about 0.54 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.39 mg/kg or about 0.54 mg/kg of the immunoconjugate is administered.

[0020] In one embodiment, about 0.45 mg/kg of the immunoconjugate is administered.

In one embodiment, about 0.5 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.55 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.6 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.65 mg/kg of the immunoconjugate is administered. In one embodiment, about 0.7 mg/kg of the immunoconjugate is administered.

[0021] In one embodiment, the immunoconjugate is administered about once a week.

[0022] In one embodiment, the immunoconjugate is administered on a 28-day cycle.

[0023] In one embodiment, wherein the immunoconjugate is administered by intravenous infusion.

[0024] In one embodiment, the variable heavy chain comprises the sequence set forth in

SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10.

[0025] In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0026] In one embodiment, the immunoconjugate is an immunoconjugate of Formula

(II):

or a pharmaceutically acceptable salt thereof, wherein A is the anti-CD33 antibody or antigen-binding fragment thereof and r is an integer from 1 to 10.

[0027] In one embodiment, the immunoconjugate is an immunoconjugate of Formula

(IIΙ):

or a pharmaceutically acceptable salt thereof wherein A is the anti-CD33 antibody or antigen-binding fragment thereof and r is an integer from 1 to 10.

[0028] In one embodiment, the pharmaceutically acceptable salt is a sodium or potassium salt.

[0029] In one embodiment, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6.

[0030] In one embodiment, the immunoconjugate is contained in a composition

comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5.

[0031] In one embodiment, the indolino-benzodiazepine dimer (e.g., DGN462) is linked to the anti-CD33 antibody or antigen-binding fragment thereof via a lysine residue of the antibody or antigen-binding fragment thereof. In one embodiment, the 1-10, 2-9, 3-8, 4-7, or 5-6 indolino-benzodiazepine dimer molecules (e.g., DGN462) are attached to the anti- CD33 antibody or antigen-binding fragment thereof via lysine residues of the antibody or antigen-binding fragment thereof. In one embodiment, the 2-8, 2-7, 3-5, or 2.5-3.5 indolino-benzodiazepine dimer molecules (e.g., DGN462) are attached to the anti-CD33 antibody or antigen-binding fragment thereof via lysine residues of the antibody or antigen-binding fragment thereof.

[0032] In one embodiment, the immuoconjugate is IMGN779.

[0033] In one embodiment, the cancer is selected from the group consisting of leukemia, lymphoma and myeloma. In one embodiment, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), T- cell acute lymphoblastic leukemia (T ALL) chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In one embodiment, the cancer is acute myeloid leukemia (AML).

[0034] In one embodiment, the AML is refractory or relapsed acute myeloid leukemia. In one embodiment, the AML is characterized by overexpression of P-glycoprotein, overexpression of EVI1, a p53 alteration, DNMT3A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2.

[0035] In one embodiment, a homozygous rsl2459419C genotype has been detected in sample obtained from the subject. In one embodiment, the method further comprises detecting the homozygous rsl2459419C genotype in a sample obtained from the subject.

[0036] In one embodiment, a heterozygous rsl2459419C genotype has been detected in sample obtained from the subject. In one embodiment, the method further comprises detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject.

[0037] In one embodiment, a homozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one embodiment, a method of treating cancer comprises detecting the homozygous rsl2459419T genotype in a sample obtained from the subject.

[0038] In one embodiment, a heterozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one embodiment, a method of treating cancer comprises detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject.

[0039] In one embodiment, the sample obtained from the subject is a blood sample or a buccal swab.

[0040] In one embodiment, the cancer is characterized by a RAS mutation. In one

embodiment, the cancer is characterized by a TP53 mutation. In one embodiment, the cancer is characterized by a IDH mutation. In one embodiment, the cancer is

characterized by an FLT3 mutation.

[0041] In one embodiment, the cancer is chemotherapy sensitive.

[0042] In one embodiment, the cancer is chemotherapy resistant.

[0043] In one embodiment, at least 20% of blasts from the cancer are CD33 -positive as measured by flow cytometry. [0044] In one embodiment, the anti-CD33 immunoconjugate (e.g., IMGN779) is administered at a dose (e.g., 0.39 mg/kg or 0.54 mg/kg; e.g., weekly or once per two weeks) that maintains exposure to the anti-CD33 immunoconjugate e.g., at least through seven days from infusion.

[0045] In one embodiment, the administration results in saturation of free-CD33.

[0046] In one embodiment, the administration results in a decrease in peripheral blood blasts, e.g., within 3-8 days of the first dose or after the second dose of the anti-CD33 immunoconjugate (e.g., IMGN779).

[0047] In one embodiment, the administration results in a decrease in bone marrow blasts

(e.g., a decrease from baseline of at least 45%, at least 48%, at least 59%, or at least

96%).

[0048] In one embodiment, the subject is a human.

[0049] In one embodiment, a method of treating AML in a human comprises

administering about once in two weeks to the subject about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0 mg/kg of an anti-CD33

immunoconjugate of formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H; r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0050] In one embodiment, a method of treating AML in a human comprises

administering about once in two weeks to the subject about 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, or 1.25 mg/kg of an anti-CD33 immunoconjugate of formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H; r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising the sequence set forth in SEQ ID NO: 1 1 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0051] In one embodiment, a method of treating AML in a human comprises

administering about once in two weeks to the subject about 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, or 2.0 mg/kg of an anti-CD33 immunoconjugate of formula:

[0052] In one embodiment, a method treating AML in a human comprises administering weekly to the subject about 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or 0.7 mg/kg of an anti-CD33 immunoconjugate of formula:

[0053] In one embodiment, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6.

[0054] In one embodiment, the indolino-benzodiazepine dimer (e.g., DGN462) is linked to the anti-CD33 antibody or antigen-binding fragment thereof via a lysine residue of the antibody or antigen-binding fragment thereof. In one embodiment, the 1-10, 2-9, 3-8, 4-7, or 5-6 indolino-benzodiazepine dimer molecules (e.g., DGN462) are attached to the anti- CD33 antibody or antigen-binding fragment thereof via lysine residues of the antibody or antigen-binding fragment thereof.

[0055] In one instance (II), a method of treating cancer in a subject comprises

administering to the subject about 0.54 to about 0.8 mg/kg of an anti-CD33

immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H; r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0056] In one instance (12) of II, about 0.54 to about 0.70 mg/kg of the immunoconjugate is administered. In one instance (13) of II, about 0.54 or about 0.70 mg/kg of the immunoconjugate is administered.

[0057] In one instance (14) of any one of 11-13, the immunoconjugate is administered about once in two weeks.

[0058] In one instance (15), a method of treating cancer in a subject comprises

administering to the subject about 0.30 to about 0.54 mg/kg of an anti-CD33

immunoconjugate of the following formula:

[0059] In one instance (16) of 15, about 0.39 to about 0.54 mg/kg of the immunoconjugate is administered. In one instance (17) of 15, about 0.39 or about 0.54 mg/kg of the immunoconjugate is administered. In one instance (18) of any one of 15-17, the immunoconjugate is administered about once a week.

[0060] In one instance (19) of any one of 11-18, the immunoconjugate is administered on a

28-day cycle. In one instance (110) of any one of 11-19, the immunoconjugate is administered by intravenous infusion. In one instance (II 1) of any one of 11-110, the variable heavy chain comprises the sequence set forth in SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10. In one instance (112) of II 1, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0061] In one instance (113) of any one of 11-112, the immunoconjugate is an

immunoconjugate of Formula (Π):

[0062] In one instance (114) of any one of 11-112, the immunoconjugate is an

immunoconjugate of Formula (ΠΙ):

[0063] In one instance (II 5) of any one of 11-114, the pharmaceutically acceptable salt is a sodium or potassium salt. In one instance (116) of any one of 11-115, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6. In one instance (117) of any one of 11-115, the immunoconjugate is contained in a composition comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5. In one instance (118) of any one of 11-117, the immunoconjugate is IMGN779.

[0064] In one instance (119) of any one of 11-118, the cancer is selected from the group consisting of leukemia, lymphoma and myeloma. In one instance (120) of 119, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute

lymphoblastic leukemia (B ALL), T-cell acute lymphoblastic leukemia (T ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplasia syndrome (MDS), basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In one instance (121) of 120, the cancer is acute myeloid leukemia (AML). In one instance (122) of 121, the AML is refractory or relapsed acute myeloid leukemia. In one instance (123) of 121 or 122, the AML is characterized by overexpression of P-glycoprotein, overexpression of EVTl, a p53 alteration, D MT3 A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2.

[0065] In one instance (124) of any one of 11-123, a homozygous rsl2459419C genotype has been detected in a sample obtained from the subject. In one instance (125) of 124, the method further comprises detecting the homozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (126) of any one of 11-123, a heterozygous rsl2459419C genotype has been detected in a sample obtained from the subject. In one instance (127) of 126, the method further comprises detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (128) of any one of 11-123, a homozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one instance (129) of 128, the method further comprises detecting the homozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (130) of any one of 11-123, a heterozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one instance (131) of claim 130, the method further comprises detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (132) of any one of 124-131, the sample obtained from the patient is a blood sample or a buccal swab.

[0066] In one instance (133) of any one of 11-132, the cancer is chemotherapy sensitive.

In one instance (134) of any one of 11-132, the cancer is chemotherapy resistant. In one instance (135) of any one of 11 -134, at least 20% of blasts from the cancer are CD33- positive as measured by flow cytometry.

[0067] In one instance (136) of any one of 11-135, the administration results in saturation of free-CD33.

[0068] In one instance (137) of any one of 11-136, the subject is a human.

[0069] In one instance (138), a method of treating AML in a human comprises

administering about once in two weeks to the subject about 0.7 mg/kg of an anti-CD33 immunoconjugate of formula

[0070] In one instance (139), a method of treating AML in a human comprising

administering weekly to the subject about 0.54 mg/kg of an anti-CD33 immunoconjugate of formula

[0071] In one instance (140) of 138 or 139, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6.

[0072] In one instance (141) a method of treating cancer in a subject comprises

administering to the subject about 0.7 to about 1 mg/kg of an anti-CD33

immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H; r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively and a variable light chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively.

[0073] In one instance (142) of 141, about 0.7 to about 0.9 mg/kg of the immunoconjugate is administered. In one instance (143) of 141, about 0.7 mg/kg of the immunoconjugate is administered. In one instance (144) of 141, about 0.75 mg/kg of the immunoconjugate is administered. In one instance (145) of 141, about 0.8 mg/kg of the immunoconjugate is administered. In one instance (146) of 141, about 0.85 mg/kg of the immunoconjugate is administered. In one instance (147) of 141, about 0.9 mg/kg of the immunoconjugate is administered. In one instance (148) of 141, about 0.95 mg/kg of the immunoconjugate is administered. In one instance (149) of 141, about 1 mg/kg of the immunoconjugate is administered. In one instance (150) of any one of 141-149, the immunoconjugate is administered about once in two weeks.

[0074] In one instance (151), a method of treating cancer in a subject comprises

administering to the subject about 0.39 to about 0.7 mg kg of an anti-CD33

immunoconjugate of the following formula:

[0075] In one instance (152) of 151 about 0.39 mg/kg of the immunoconjugate is

administered. In one instance (153) of 151, about 0.45 mg/kg of the immunoconjugate is administered. In one instance (154) of 151, about 0.5 mg/kg of the immunoconjugate is administered. In one instance (155) of 151, about 0.54 mg/kg of the immunoconjugate is administered. In one instance (156) of 151, about 0.55 mg/kg of the immunoconjugate is administered. In one instance (157) of 151, about 0.6 mg/kg of the immunoconjugate is administered. In one instance (158) of 151, about 0.65 mg/kg of the immunoconjugate is administered. In one instance (159) of 151, about 0.7 mg/kg of the immunoconjugate is administered. In one instance (160) of any one of 151-159, the immunoconjugate is administered about once a week.

[0076] In one instance (161) of any one of 141-160, the immunoconjugate is administered on a 28-day cycle. In one instance (162) of any one of 141-161, the immunoconjugate is administered by intravenous infusion. In one instance (163) of any one of 141-162, the variable heavy chain comprises the sequence set forth in SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10. In one instance (164) of 163, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12. In one instance (165) of any one of 141-164, the immunoconjugate is an immunoconjugate of Formula (Π):

[0077] In one instance (166) of any one of 141-164, the immunoconjugate is an

immunoconjugate of Formula (III):

[0078] In one instance (167) of any one of 141-166, the pharmaceutically acceptable salt is a sodium or potassium salt. In one instance (168) of any one of 141-167, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6. In one instance (169) of any one of 141-167, the immunoconjugate is contained in a composition comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5. In one instance (170) of any one of 141-169, the immuoconjugate is IMGN779. In one instance (171) of any one of 141-170, the cancer is selected from the group consisting of leukemia, lymphoma and myeloma. In one instance (172) of 171, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), T-cell acute lymphoblastic leukemia (T ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDC ) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In one instance (173) of 172, the cancer is acute myeloid leukemia (AML). In one instance (174) of 173, the AML is refractory or relapsed acute myeloid leukemia. In one instance (175) of 173, the AML is newly diagnosed AML. In one instance (176) of any one of 171-175, the AML is characterized by overexpression of P-glycoprotein, overexpression of EVI1, a p53 alteration, DNMT3A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2. In one instance (177) of 172, the MDS is high risk MDS.

[0079] In one instance (178) of any one of 141-77, a homozygous rsl2459419C genotype has been detected in a sample obtained from the subject. In one instance (179) of 178, the method further comprises detecting the homozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (180) of any one of 141-177, a heterozygous rsl2459419C genotype has been detected in a sample obtained from the subject. In one instance (181) of 180, the method further comprises detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (182) of any one of 141-177, a homozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one instance (183) of 182, the method further comprises detecting the homozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (184) of any one of 141-177, a heterozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one instance (185) of 184, the method further comprises detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (186) of any one of 178-185, the sample obtained from the patient is a blood sample or a buccal swab.

[0080] In one instance (187) of any one of 141-186, the cancer is chemotherapy sensitive.

In one instance (188) of any one of 141-186, the cancer is chemotherapy resistant. In one instance (189) of any one of 141-188, at least 20% of blasts from the cancer are CD33- positive as measured by flow cytometry. In one instance (190) of any one of 141-189, the administration results in saturation of free-CD33.

[0081] In one instance (191) of any one of 141-190, the administration results in a

decrease in peripheral blood blasts. In one instance (192) of any one of 141-191, the administration results in a decrease in bone marrow blasts. In one instance (193) of any one of 141-190, the subject is a human.

[0082] In one instance (194), a method of treating cancer in a subject comprises

administering to the subject about 0.7 to about 1.25 mg/kg of an anti-CD33

immunoconjugate of the following formula:

[0083] In one instance (195) of 194, about 0.7 to about 1 mg/kg of the immunoconjugate is administered. In one instance (196) of 194, about 0.7 to about 0.9 mg/kg of the immunoconjugate is administered. In one instance (197) of 194, about 0.7 mg/kg of the immunoconjugate is administered. In one instance (198) of 194, about 0.75 mg/kg of the immunoconjugate is administered. In one instance (199) of 194, about 0.8 mg/kg of the immunoconjugate is administered. In one instance (1100) of 194, about 0.85 mg/kg of the immunoconjugate is administered. In one instance (1101) of 194, about 0.9 mg/kg of the immunoconjugate is administered. In one instance (1102) of 194, about 0.95 mg/kg of the immunoconjugate is administered. In one instance (1103) of 194 about 1 mg/kg of the immunoconjugate is administered.

[0084] In one instance (1104) of any one of 194-1103, the immunoconjugate is

administered about once a week.

[0085] In one instance (1105) of 194 about 1 to about 1.25 mg/kg of the immunoconjugate is administered. In one instance (1106) of 194, about 1.1 mg/kg of the immunoconjugate is administered. In one instance (1107) of 194, about 1.15 mg/kg of the immunoconjugate is administered. In one instance (1108) of 194, about 1.2 mg/kg of the immunoconjugate is administered. In one instance (1109) of 194, about 1.25 mg/kg of the immunoconjugate is administered. In one instance (II 10) of any one of 194 and 1105-1109, the

immunoconjugate is administered about once in two weeks. In one instance (II 11) of any one of 194 and 1105-1109 the immunoconjugate is administered about once a week.

[0086] In one instance (II 12) of any one of 194-1111, the immunoconjugate is

administered on a 28-day cycle. In one instance (II 13) of any one of 194-1112, the immunoconjugate is administered by intravenous infusion. In one instance (II 14) of any one of 194-11 13, the variable heavy chain comprises the sequence set forth in SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10. In one instance (II 15) of II 14, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0087] In one instance (1116) of any one of 194-1115, the immunoconjugate is an

immunoconjugate of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein A is the anti-CD33 antibody or antigen-binding fragment thereof and r is an integer from 1 to 10. [0088] In one instance (1117) of any one of 194-1115, the immunoconjugate is an immunoconjugate of Formula (III):

[0089] In one instance (II 18) of any one of 194-117, the pharmaceutically acceptable salt is a sodium or potassium salt. In one instance (II 19) of any one of 194-1118, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6. In one instance (1120) of any one of 194-1118, the immunoconjugate is contained in a composition comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5. In one instance (1121) of any one of 194-1120, the immunoconjugate is EVIGN779.

[0090] In one instance (1122) of any one of 194-1121 the cancer is selected from the group consisting of leukemia, lymphoma and myeloma. In one instance (1123) of 1122, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), T-cell acute lymphoblastic leukemia (T ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In one instance (1124) of 1123, the cancer is acute myeloid leukemia (AML). In one instance (1125) of 1124, the AML is refractory or relapsed acute myeloid leukemia. In one instance (1126) of 1124, the AML is newly diagnosed AML. In one instance (1127) of any one of 1124-1126, the AML is characterized by overexpression of P-glycoprotein, overexpression of EVI1, a p53 alteration, DNMT3 A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2. In one instance (1128) of 1123, the MDS is high risk MDS.

[0091] In one instance (1129) of any one of 194-1128, a homozygous rsl2459419C

genotype has been detected in a sample obtained from the subject. In one instance (1130) of 1129, the method further comprises detecting the homozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (1131) of any one of 194-1128, a heterozygous rsl2459419C genotype has been detected in a sample obtained from the subject. In one instance (1132) of 1131, the method of further comprises detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (1133) of any one of 194-1128, a homozygous rsl2459419T genotype has been detected in sample obtained from the subject. In one instance (1134) of 1133, the method further comprises detecting the homozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (1135) of anyone of 194-1128, a heterozygous rsl2459419T genotype has been detected in a sample obtained from the subject. In one instance (1136) of 1135, the method further comprises detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (1137) of any one of 1129-1136, the method of any one of claims 129-136, the sample obtained from the patient is a blood sample or a buccal swab.

[0092] In one instance (1138) of any one of 194-1137, the cancer is chemotherapy

sensitive. In one instance (1139) of any one of 194-1137, the cancer is chemotherapy resistant. In one instance (1140) of any one of 194-1139, at least 20% of blasts from the cancer are CD33-positive as measured by flow cytometry.

[0093] In one instance (1141) of any one of 194-1140, the administration results in

saturation of free-CD33. In one instance (1142) of any one of 194-1141, the

administration results in a decrease in peripheral blood blasts. In one instance (1143) of any one of 194-1142, the administration results in a decrease in bone marrow blasts. In one instance (1144) of any one of 194-1143, the subject is a human.

[0094] In one instance (1145), a method of treating cancer comprises administering to the subject about 1 to about 2 mg/kg of an anti-CD33 immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein: the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H;r is an integer from 1 to 10; and A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively and a variable light chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively.

[0095] In one instance (1146) of 1145, about 1.2 to about 2 mg/kg of the

immunoconjugate is administered. In one instance (1147) of 1145, about 1.5 to about 1.6 mg/kg mg/kg of the immunoconjugate is administered. In one instance (1148) of 1145, about 1.5 mg/kg or about 1.56 mg/kg of the immunoconjugate is administered. In one instance (1149) of any one of 1145-1148, the immunoconjugate is administered about once in two weeks. In one instance (1150) of any one of 1145-1148, the immunoconjugate is administered about once a week.

[0096] In one instance (1151) of any one of 1145-1150, the immunoconjugate is

administered on a 28-day cycle. In one instance (1152) of any one of 1145-1151, the immunoconjugate is administered by intravenous infusion. In one instance (1153) of any one of 1145-1152, the variable heavy chain comprises the sequence set forth in SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10. In one instance (1154) of 1153, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

[0097] In one instance (1155) of any one of 1145-1154, the immunoconjugate is an

immunoconjugate of Formula (II):

[0098] In one instance (I156) of any one of 1145-1154, the immunoconjugate is an

immunoconjugate of Formula (IIΙ):

[0099] In one instance (I 157) of any one of 1145-1156, the pharmaceutically acceptable salt is a sodium or potassium salt. In one instance (1158) of any one of 1145-1157, r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6. In one instance (1159) of any one of 1145-1157, the immunoconjugate is contained in a composition comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5. In one instance (1160) of any one of 1145-1159, the immuoconjugate is IMGN779.

[0100] In one instance (1161) of any one of 1145-1160, the cancer is selected from the group consisting of leukemia, lymphoma and myeloma. In one instance (1162) of 1161 , the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), T-cell acute lymphoblastic leukemia (T ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDC ) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In one instance (1163) of 1162, the cancer is acute myeloid leukemia (AML). In one instance (1164) of 1163, the AML is refractory or relapsed acute myeloid leukemia. In one instance (1165) of 1163, the AML is newly diagnosed AML. In one instance (1166) of any one of 1163-165, the AML is characterized by overexpression of P-glycoprotein, overexpression of EVI1, a p53 alteration, DNMT3A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2. In one instance (1167) of 1162, the MDS is high risk MDS.

[0101] In one instance (1168) of any one of 1145-1167, a homozygous rsl2459419C

genotype has been detected in sample obtained from the subject. In one instance (1169) of 1168, the method further comprises detecting the homozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (1170) of any one of 1145-1167, a heterozygous rsl2459419C genotype has been detected in sample obtained from the subject. In one instance (1171) of 1170, the method further comprises detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject. In one instance (1172) of any one of 1145-1167, a homozygous rsl2459419T genotype has been detected in sample obtained from the subject. In one instance (1173) of II 72, the method further comprises detecting the homozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (1174) of any one of 1145-1167, a heterozygous rsl2459419T genotype has been detected in sample obtained from the subject. In one instance (1175) of 1174, the method further comprises detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject. In one instance (1176) of any one of 1168 to 1175, the sample obtained from the patient is a blood sample or a buccal swab.

[0102] In one instance (1177) of any one of 1145-1176, the cancer is chemotherapy

sensitive. In one instance (1178) of any one of 1145-1176, the cancer is chemotherapy resistant. In one instance (1179) of any one of 1145-1178, at least 20% of blasts from the cancer are CD33 -positive as measured by flow cytometry. [0103] In one instance (1180) of any one of 1145-1179, the administration results in saturation of free-CD33. In one instance (1181) of any one of 1145-1180, the

administration results in a decrease in peripheral blood blasts. In one instance (1182) of any one of 1145-1181, the administration results in a decrease in bone marrow blasts. In one instance (1183) of any one of 1145-1182, the subject is a human.

[0104] In one instance (1184) of any one of 11-1183, the cancer is chacterized by a RAS mutation. In one instance (1185) of any one of 11-1184, the cancer is chacterized by a TP53 mutation. In one instance (1186) of any one of 11-185, the cancer is chacterized by an IDH mutation. In one instance (1187) of any one of 11 -1186, the cancer is chacterized by an FLT3 mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1A shows the in vitro potency of IMGN779 in EOL-1 cell line.

[0106] FIG. IB shows the in vitro potency of IMGN779 in MV4-11 cell line.

[0107] FIG. 2A shows the in vitro potency of DGN462 and DGN484 in three AML cell lines.

[0108] FIG. 2B shows the apoptotic activity of DGN462 and DGN484 in three AML cell lines.

[0109] FIG. 3 shows the indirect binding of CD33 antibody drug conjugates against

MOLM-13 cells.

[0110] FIG. 4A shows the in vitro potency of IMGN779 and Z4681A-sSPDB-DGN484 in MOLM-13 cell line.

[0111] FIG. 4B shows the in vitro potency of IMGN779 and Z4681A-sSPDB-DGN484 in

MV4-11 cell line.

[0112] FIG. 4C shows the in vitro potency of IMGN779 and Z4681A-sSPDB-DGN484 in

EOL-1 cell line.

[0113] FIG. 4D shows the in vitro potency of IMGN779 and Z4681A-sSPDB-DGN484 in HNT-34.

[0114] FIG. 5 shows the results of an intravenous single dose tolerability study of

IMGN779 in female CD-I mice.

[0115] FIG. 6 shows the results of an intravenous single dose tolerability study of

IMGN779 and Z4681A-sSPDB-DGN484 conjugate in female CD-I mice. [0116] FIG. 7 shows the results of a single dose in vivo efficacy study of IMGN779 in an

EOL-1 subcutaneous model.

[0117] FIG. 8 shows the results of a single dose in vivo efficacy study of IMGN779 in a

HL60/QC subcutaneous model.

[0118] FIG. 9 shows the results of single and multiple dose in vivo efficacy studies of

IMGN779 in HL60/QC subcutaneous model.

[0119] FIG. 10 shows the results of a single dose in vivo efficacy study of IMGN779 and

Z4681 A-sSPDB-DGN484 in a HL60/QC subcutaneous model.

[0120] FIG. 11 shows the results of a single dose vs. multiple fractionated dose in vivo efficacy study of IMGN79 in a MV4-11 subcutaneous model.

[0121] FIG. 12 shows the results of a single dose in vivo efficacy study of IMGN779 in a

MV4-11 disseminated model.

[0122] FIG. 13 shows the results of a multiple dose in vivo efficacy study of EVIGN779 in a MV4-11 disseminated model.

[0123] FIG. 14 shows the results of a single dose in vivo efficacy study of IMGN779 and

Z6481A-sSPDB-DGN484 in a MV4-11 disseminated model.

[0124] FIG. 15 shows the results of a multiple dose in vivo efficacy study of IMGN779 in a Molm-13 disseminated model.

[0125] FIG. 16 shows the demographics of 23 patients treated with IMGN779 (0.02 mg/kg to 0.54 mg/kg).

[0126] FIG. 17 shows the treatment-emergent adverse events that occurred with a

frequency of at least 10 percent in 23 patients treated with IMGN779 (0.02 mg/kg to 0.54 mg/kg).

[0127] FIG. 18 shows the adverse events that were Grade 3 or higher and occurred in at least two patients out of 23 patients treated with IMGN779 (0.02 mg/kg to 0.54 mg/kg).

[0128] FIG. 19 shows the change from baseline (%) in absolute peripheral blast counts in patients in cohorts 6 and 7.

[0129] FIG. 20 shows the maximum change from baseline in bone marrow blasts in

patients in cohorts 6 and 7.

[0130] FIG. 21 A shows IMGN779 concentration over time at various doses of

IMGN779.

[0131] FIG. 21B shows the calculated Cmax value for Cycle 1 (first cycle of dosing for each patient) at various doses of EVIGN779. [0132] FIG. 21C shows the calculated AUC for Cycle 1 (first cycle of dosing for each patient) at various doses of IMGN779.

[0133] FIG. 22 shows the concentration of IMGN779 (left panel) and the CD33

saturation (right panel) in patients in cohorts 4-7.

[0134] FIG. 23 shows the IMGN779 exposure (left panel) and the CD33 saturation (right panel) in patients treated with 0.04 to 0.91 mg/kg EVIGN779.

[0135] FIG. 24 shows the CD33 saturation (left) and end of infusion concentration (right) in patients treated with 0.39 mg/kg IMGN779 once weekly (QW) or once every two weeks (Q2W).

[0136] FIG. 25 shows the percentage change in bone marrow blasts in patients treated with IMGN779.

[0137] FIG. 26 shows the Grade 3+ treatment-emergent adverse events (TEAEs)

occurring in more than 1 patient following administration of IMGN779.

[0138] FIGs. 27A and 27B show the concentration of IMGN779 in patients' blood after receiving various doses. FIG. 27A shows the concentration of ADC (EVIGN779) throughout Cycle 1 (two weeks) for various doses, and FIG. 27B shows the end of infusion IMGN779 concentrations in various patients associated with dosing at 0.39 mg/kg Q2W and 0.39 mg/kg QW.

[0139] FIGs. 28A and 28B show the pharmacodynamics associated with CD33 saturation at various doses. FIG. 28A shows the CD33 receptor availability in blasts from patients treated with various concentrations of IMGN779 on a Q2W schedule, and FIG. 28B shows the CD33 receptor availability in blasts from patients treated with various concentrations of IMGN779 on a QW schedule.

[0140] FIGs. 29A and 29B show a comparison of Q2W and QW dosing schedules at a dose of 0.54 mg/kg. FIG. 29 A shows the CD33 receptor availability in blasts from patients treated with 0.54 mg/kg EVIGN779 on a Q2W schedule, and FIG. 29B shows the CD33 receptor availability in blasts from patients treated with 0.54 mg/kg IMGN779 on a QW schedule.

[0141] FIGs. 30A and 30B show CD33 expression in leukemic cells obtained from

patients treated with IMGN779. FIG. 30A shows CD33 levels plotted against binding sites per cell in patients from Cohort 6+, and FIG. 30B shows the percent CD33+ leukemic cells in local site bone marrow. Asterisks indicate formal or non-formal bone marrow responses (>25% decrease in bone marrow blasts + clearance of peripheral blood (PB)).

[0142] FIG. 31 shows the average CD33 antigens per cell for patients with three different

SNP allele combinations. Asterisks indicate formal or non-formal bone marrow responses

(>25% decrease in bone marrow blasts + clearance of PB).

[0143] FIG. 32 shows the best response obtained in patients treated with various dosing schedules of IMGN779.

[0144] FIG. 33 shows the elimination rate constant estimates obtained from elimination half-life data on plasma ADC (TMGN779) at various doses.

[0145] FIG. 34 shows that a linear pharmacokinetic (PK) model does not fit IMGN779

PK data.

[0146] FIGs. 35 A and 35B show the IMGN779 elimination rate constant associated with the number of binding sites on a target cell (FIG. 35 A) and with the total target concentration (FIG. 35B).

[0147] FIG. 36 shows that a target-mediated drug disposition model does not fit

IMGN779 PK data.

[0148] FIGs. 37A and 37B show a scatter plot (FIG. 37A) and a box plot (FIG. 37B) for the measured target and estimated target in patients treated with IMGN779.

[0149] FIG. 38 shows that a binding model is a better fit for IMGN779 PK data.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0150] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0151] The term "CD33," as used herein, refers to any native CD33 polypeptide, unless otherwise indicated. The term "CD33" encompasses "full-length," unprocessed CD33 polypeptide as well as any forms of CD33 that result from processing within the cell (e.g., removal of the signal peptide). The term also encompasses naturally occurring variants of CD33, e.g., those encoded by splice variants and allelic variants. The CD33 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Where specifically indicated, "CD33" can be used to refer to a nucleic acid that encodes a CD33 polypeptide. Human CD33 sequences are known and include, for example, the sequences publically available at NCBI Accession No. CAD36509 (including isoforms). As used herein, the term "human CD33" refers to CD33 comprising the sequence of amino acids 18-364 of SEQ ID NO: 14:

1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctf fhpipyy dknspvhgyw

61 fregaiisrd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr rdngsyffrm

121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace qgtppifswl

181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln vtyvpqnptt

241 gifpgdgsgk qetragvvhg aiggagvtal lalclcliff ivkthrrkaa rtavgrndth

301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp skdtsteyse

361 vrtq ( SEQ ID NO : 14 )

[0152] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0153] The term "antibody fragment" refers to a portion of an intact antibody. An

"antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. Antibody fragments can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0154] The term "anti-CD33 antibody" or "an antibody that binds to CD33" refers to an antibody that is capable of binding CD33 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD33. The extent of binding of an anti-CD33 antibody to an unrelated, non-CD33 protein can be less than about 10% of the binding of the antibody to CD33 as measured, e.g., by a radioimmunoassay (RIA).

[0155] A "monoclonal" antibody or antigen-binding fragment thereof refers to a

homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen- binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore,

"monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0156] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability ("CDR grafted") (Jones et al., Nature 321 :522-525 (1986);

Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody or antigen-binding fragment thereof specificity, affinity, and/or capability. In general, the humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969- 973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some

embodiments, a "humanized antibody" is a resurfaced antibody.

[0157] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.), "Kabat"); and (2) an approach based on

crystallographic studies of antigen-antibody complexes (Al-lazikani et al, J. Molec. Biol. 273:927-948 (1997)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0158] A "constant" region of an antibody is not involved directly in binding an antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity

[0159] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed., 1991, National Institutes of Health, Bethesda, Md.) ("Kabat").

[0160] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al. (Sequences of Immunological Interest. 5th Ed., 1991, National Institutes of Health, Bethesda, Md.), ("Kabat"). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.

residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[0161] The term "human" antibody or antigen-binding fragment thereof means an

antibody or antigen-binding fragment thereof produced by a human or an antibody or antigen-binding fragment thereof having an amino acid sequence corresponding to an antibody or antigen-binding fragment thereof produced by a human made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0162] The term "chimeric" antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0163] The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0164] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0165] "Or better" when used herein to refer to binding affinity refers to a stronger

binding between a molecule and its binding partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

[0166] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."

[0167] By "preferentially binds," it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0168] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0169] The phrase "substantially similar," or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.

[0170] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is

"isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0171] As used herein, "substantially pure" refers to material which is at least 50% pure

(i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0172] The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e., an anti-CD33 antibody or antigen-binding fragment thereof) and is defined by a generic formula: C-L-A, wherein C = cytotoxin, L = linker, and A = anti-CD33 antibody or antibody fragment.

Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.

[0173] A "linker" is any chemical moiety that is capable of linking a compound, usually a drug, such as an indolino-benzodiazepine dimer, to a cell-binding agent such as an anti CD33 antibody or antigen-binding fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.

Suitable linkers are well known in the art and include, for example, disulfide groups and thioether groups.

[0174] The term "IMGN779" refers to a CD33-targeted ADC comprising the huMy9-6 antibody (i.e., an antibody comprising the heavy chain CDRl-3 having the sequence of SEQ ID NOs: 1-3, respectively and the light chain CDRl-3 having the sequence of SEQ ID NOs:4-6; comprising the heavy chain variable region having the sequence of SEQ ID NO:9 and a light chain variable region having the sequence of SEQ ID NO: 10;

comprising the heavy chain sequence having the sequence of SEQ ID NO: 11 and the light chain sequence having the sequence of SEQ ID NO: 12), conjugated to DGN462, via the cleavable disulfide linker N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo- SPDB). The huMy9-6 antibody is also known as the Z4681A antibody. IMGN779 is formulated as a combination of Formulas IV and V depicted below:

or a pharmaceutically acceptable salt thereof; and

or a pharmaceutically acceptable salt thereof.

[0175] The terms "cancer" and "cancerous" refer to or describe the physiological

condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). The cancer can be a cancer that expresses CD33.

[0176] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells. [0177] An "advanced" cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis. The term "advanced" cancer includes both locally advanced and metastatic disease.

[0178] "Metastatic" cancer refers to cancer that has spread from one part of the body ) to another part of the body.

[0179] A "refractory" cancer is one that progresses even though an anti-tumor treatment, such as a chemotherapy, is administered to the cancer patient.

[0180] A "recurrent" cancer is one that has regrown, either at the initial site or at a distant site, after a response to initial therapy.

[0181] A "relapsed" patient is one who has signs or symptoms of cancer after remission.

Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0182] The terms "line of treatment" or "line of therapy" refer to a therapeutic regimen that can include but is not limited to surgery, radiation therapy, chemotherapy, differentiating therapy, biotherapy, immune therapy, or the administration of one or more anti-cancer agents (e.g., a cytotoxic agent, an anti-proliferative compound, and/or an angiogenesis inhibitor).

[0183] The terms "first-line treatment," "first-line therapy," and "front-line therapy" refer to the preferred and standard initial treatment for a particular condition, e.g., a given type and stage of cancer. These treatments differ from "second-line" therapies, which are tried when a first-line therapy does not work adequately. "Third-line" therapies are tried when a first-line therapy and a second-line therapy do not work adequately.

[0184] The term "maintenance therapy" refers to therapy that is given to help keep cancer from coming back after it has disappeared following the initial therapy.

[0185] The term "unfit AML" as used herein refers to a subject having AML who is

ineligible for intensive therapy. The measures for determining a subject with unfit AML include, e.g., physical performance (as determined by e.g., the Eastern Cooperative Oncology Group performance status (ECOG PS), the Karnofsky performance status (KPS), and the short physical performance battery (SPPB)), comorbid conditions (as determined by the Charlson comorbidity index (CCI) or the hematopoietic cell transplantation-specific comorbidity index (HCT-CI)), cognitive function, and prognostic models (including but not limited to, cytogenetic group, age, white blood cell count, LDH, type of AML). In some cases, an unfit AML subject is a subject over the age of 60. [0186] The term "fit AML" as used herein refers to a subject having AML who is eligible for intensive therapy. The measures for determining a subject with fit AML include, e.g., physical performance (as determined by e.g., the Eastern Cooperative Oncology Group performance status (ECOG PS), the Karnofsky performance status (KPS), and the short physical performance battery (SPPB)), comorbid conditions (as determined by the Charlson comorbidity index (CCI) or the hematopoietic cell transplantation-specific comorbidity index (HCT-CI)), cognitive function, and prognostic models (including but not limited to, cytogenetic group, age, white blood cell count, LDH, type of AML). In some cases, a fit AML subject is a subject at the age of 60 or under the age of 60.

[0187] The term "P-glycoprotein" as used herein refers to any native P-glycoprotein, unless otherwise indicated. The term "P-glycoprotein" encompasses "full-length," unprocessed P-glycoprotein as well as any form of P-glycoprotein that results from processing within the cell. The term also encompasses naturally occurring variants of P- glycoprotein, e.g., those encoded by splice variants and allelic variants. The P- glycoproteins described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Where specifically indicated, " P-glycoprotein" can be used to refer to a nucleic acid that encodes a P-glycoprotein. Human P-glycoprotein sequences are known and include, for example, the sequences publically available at NCBI Accession No.

NP 001035830 (including isoforms). As used herein, the term "human P-glycoprotein" refers to P-glycoprotein comprising the sequence of SEQ ID NO: 15: .

1 maaaeaggdd arcvrlsaer aqalladvdt llfdcdgvlw rgetavpgap ealralrarg

61 krlgfitnns sktraayaek lrrlgfggpa gpgaslevfg tayctalylr qrlagapapk

121 ayvlgspala aeleavgvas vgvgpeplqg egpgdwlhap lepdvravw gfdphfsymk

181 ltkalrylqq pgcllvgtnm dnrlplengr fiagtgclvr avemaaqrqa diigkpsrfi

241 fdcvsqeygi npertvmvgd rldtdillga tcglktiltl tgvstlgdvk nnqesdcvsk

301 kkmvpdfyvd siadllpalq g ( SEQ ID NO : 15 ) .

[0188] The term "FLT3 protein," "FLT3 polypeptide," "FLT3," "FLT-3 Receptor," or

"FLT-3R" as used herein refers to any native FLT3 protein, unless otherwise indicated. The term "FLT3" encompasses "full-length," unprocessed FLT3 as well as any form of FLT3 that results from processing in the cell. The term also encompasses naturally occurring variants of FLT3, e.g., those encoded by splice variants and allelic variants. The FLT3 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Where specifically indicated, "FLT3" can be used to refer to a nucleic acid that encodes a FLT3 polypeptide. Human FLT3 sequences are known and include, for example, the sequences publically available at NCBI Accession No. NP_004110

(including isoforms). As used herein, the term "human FLT3" refers to FLT3 comprising the sequence of SEQ ID NO: 16:

1 mpalardggq lpllvvfsam ifgtitnqdl pvikcvlinh knndssvgks ssypmvsesp

61 edlgcalrpq ssgtvyeaaa vevdvsasit lqvlvdapgn iscl vfkhs slncqphfdl

121 qnrgvvsmvi lkmtetqage yllfiqseat nytilftvsi rntllytlrr pyfrkmenqd

181 alvcisesvp epivewvlcd sqgesckees pavvkkeekv lhelfgtdir ccarnelgre

241 ctrlftidln qtpqttlpql flkvgeplwi rckavhvnhg fgltwelenk aleegnyfem

301 stystnrtmi rilfafvssv arndtgyytc ssskhpsqsa lvtivekgfi natnssedye

361 idqyeefcfs vrfkaypqir ctwtfsrksf pceqkgldng ysiskfcnhk hqpgeyifha

421 enddaqftkm ftlnirrkpq vlaeasasqa scfsdgyplp swtwkkcsdk spncteeite

481 gv nrkanrk vfgqwvssst lnmseaikgf lvkccaynsl gtscetilln spgpfpfiqd

541 nisfyatigv cllfivvltl lichkykkqf ryesqlqmvq vtgssdneyf yvdfreyeyd

601 lkwefprenl efgkvlgsga fgkvmnatay gisktgvsiq vavkmlkeka dsserealms

661 elkmmtqlgs henivnllga ctlsgpiyli feyccygdll nylrskrekf hrtwteifke

721 hnfsfyptfq shpnssmpgs revqihpdsd qisglhgnsf hsedeieyen qkrleeeedl

781 nvltfedllc fayqvakgme flefkscvhr dlaarnvlvt hgkvvkicdf glardimsds

841 nyvvrgnarl pvk mapesl fegiytiksd vwsygillwe ifslgvnpyp gipvdanfyk

901 liqngfkmdq pfyateeiyi imqscwafds rkrpsfpnlt sflgcqlada eeamyqnvdg

961 rvsecphtyq nrrpfsremd lgllspqaqv eds ( SEQ ID NO : 16)

[0189] By "FLT3-ITD" is meant a FLT3 polypeptide having internal tandem

duplication(s) including but not limited to simple tandem duplication(s) and/or tandem duplication(s) with insertion. In various embodiments, FLT3 polypeptides having internal tandem duplications are activated FLT3 variants (e.g., constitutively

autophosphorylated). In some embodiments, the FLT3-ITD includes tandem duplications and/or tandem duplication(s) with insertion in any exon or intron including, for example, exon 11, exon 11 to intron 11, and exon 12, exon 14, exon 14 to intron 14, and exon 15. The internal tandem duplication mutation (FLT3-ITD) is the most common FLT3 mutation, present in about 20-25% of AML cases. Patients with FLT3-ITD AML have a worse prognosis than those with wild-type (WT) FLT3, with an increased rate of relapse and a shorter duration of response to chemotherapy.

[0190] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. [0191] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used

interchangeably herein in reference to a human subject.

[0192] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0193] An "effective amount" of an antibody, immunoconjugate, or other drug as

disclosed herein is an amount sufficient to carry out a specifically stated purpose.

[0194] The term "therapeutically effective amount" refers to an amount of an antibody, immunoconjugate, or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size or burden; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and/or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof. See the definition herein of "treating". To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.

[0195] The term "respond favorably" generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic efficacy of an anti-cancer therapeutic. With respect to tumor growth inhibition, according to NCI standards, a T/C < 42% is the minimum level of anti-tumor activity. A T/C <10% is considered a high anti-tumor activity level, with T/C (%) = Median tumor volume of the treated / Median tumor volume of the control x 100. A favorable response can be assessed, for example, by increased progression-free survival (PFS), event-free survival (EFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.

[0196] PFS, DFS, and OS can be measured by standards set by the National Cancer

Institute and the U.S. Food and Drug Administration for the approval of new drugs. See Johnson et al, (2003) J. Clin. Oncol. 21(7): 1404-1411.

[0197] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is generally measured using the Kaplan-Meier method and Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 standards. Generally, progression free survival refers to the situation wherein a patient remains alive, without the cancer getting worse.

[0198] "Event-free survival" (EFS) refers to the length of time after primary treatment ends that the patient remains free of complications or events that the treatment was intended to prevent or delay. The events may include the return of the cancer or the onset of certain symptoms.

[0199] "Time to Tumor Progression" (TTP) is defined as the time from enrollment to disease progression. TTP is generally measured using the RECIST 1.1 criteria.

[0200] A "complete response" or "complete remission" or "CR" indicates the

disappearance of all signs of tumor or cancer in response to treatment. This does not always mean the cancer has been cured.

[0201] A "complete remission with incomplete hematologic recovery" or "CRi" refers to a patient response characterized by <5% of blasts in the bone marrow but with blood counts (e.g., neutrophils and platelets) that are not within normal range.

[0202] A "partial response" or "PR" refers to a decrease in the size or volume of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.

[0203] "Stable disease" refers to disease without progression or relapse. In stable disease there is neither sufficient tumor shrinkage to qualify for partial response nor sufficient tumor increase to qualify as progressive disease.

[0204] "Progressive disease" refers to the appearance of one more new lesions or tumors and/or the unequivocal progression of existing non-target lesions. Progressive disease can also refer to a tumor growth of more than 20 percent since treatment began, either due to an increases in mass or in spread of the tumor. [0205] "Disease-free survival" (DFS) refers to the length of time during and after treatment that the patient remains free of disease.

[0206] "Overall Survival" (OS) refers to the time from patient enrollment to death or censored at the date last known alive. OS includes a prolongation in life expectancy as compared to naive or untreated individuals or patients. Overall survival refers to the situation wherein a patient remains alive for a defined period of time, such as one year, five years, etc., e.g., from the time of diagnosis or treatment. In a population of patients, overall survival is measured as mean overall survival (mOS).

[0207] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to

alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.

[0208] The terms "administer", "administering", "administration", and the like, as used herein, refer to methods that may be used to enable delivery of the immunoconjugate to the desired site of biological action. Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In one aspect, immunoconjugate is administered intravenously. [0209] The term "instructing" means providing directions for applicable therapy, medication, treatment, treatment regimens, and the like, by any means, for example, in writing, such as in the form of package inserts or other written promotional material.

[0210] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0211] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum

correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389- 3402 (1991)). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., 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). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0212] As a non-limiting example, whether any particular polynucleotide has a certain percentage sequence identity (e.g., is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482 489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0213] In some embodiments, two nucleic acids or polypeptides of the invention are

substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Identity can exist over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value there between, and can be over a longer region than 60-80 residues, for example, at least about 90-100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

[0214] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the CD33 to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);

Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0215] Unless specifically stated or obvious from context, as used herein, the term

"about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0216] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0217] As used in the present disclosure and claims, the singular forms "a," "an," and

"the" include plural forms unless the context clearly dictates otherwise.

[0218] It is understood that wherever embodiments are described herein with the

language "comprising," otherwise analogous embodiments described in terms of

"consisting of and/or "consisting essentially of are also provided. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments

[0219] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0220] Any compositions or methods provided herein can be combined with one

of any of the other compositions and methods provided herein.

//. Anti-CD33 Antibodies and Antigen-Binding Fragments Thereof

[0221] Described herein are methods of administering immunoconjugates that

specifically bind to CD33 (e.g., EVIGN779). The immunoconjugates contain an anti- CD33 antibody or antigen-binding fragment thereof. [0222] The anti-CD33 antibody can be huMy9-6 antibody.

[0223] "My9-6" (also known as "murine My9-6" and "muMy9-6") is the murine anti-

CD33 antibody from which huMy9-6 is derived. My9-6 is fully characterized with respect to the germline amino acid sequence of both light and heavy chain variable regions, amino acid sequences of both light and heavy chain variable regions, the identification of the CDRs, the identification of surface amino acids and means for its expression in recombinant form. See, for example, U.S. Patent Nos. 7,557,189;

7,342,110; 8,119,787; 8,337,855 and U.S. Patent Publication No. 20120244171, each of which is incorporated herein by reference in their entirety. The hybridoma that makes muMy9-6 antibodies was deposited with the American Type Culture Collection, PO Box 1549, Manassas, Va. 20108, on Nov. 7, 2002, under the Terms of the Budapest Treaty and was assigned deposit number PTA-4786. The amino acid sequences of muMy9-6 are also shown below in Table 1. The My9-6 antibody has also been functionally

characterized and shown to bind with high affinity to CD33 on the surface of CD33- positive cells.

Table 1

[0224] A humanized version of My9-6 is referred to herein as "huMy9-6" or "humanized

My9-6." The CDR sequences of huMy9-6 are also provided in Table 1. In order to create hyMy9-6, the CDRs of My9-6 were identified by modeling, and their molecular structures were predicted. Humanized antibodies were then prepared and have been fully characterized as described, for example in U.S. Patent Nos. 7,342,110 and 7,557,189, which are incorporated herein by reference. The amino acid sequences of the light and heavy chains of a number of huMy9-6 antibodies are described, for example, in U.S. Patent No. 8,337,855 and U.S. Patent Publication No.8,765,740, each of which is incorporated herein by reference. The variable heavy and light chain amino acid sequences, as well as the heavy and light chain amino acid sequences of huMy9-6 are provided in Table 2. Table 2

[0225] In some embodiments, the anti-CD33 immunoconjugates (e.g., IMGN779) comprise humanized antibodies or antigen-binding fragments thereof. In some embodiments, the humanized antibody or fragment is a resurfaced antibody or antigen- binding fragment thereof. In other embodiments, the anti-CD33 immunoconjugates (e.g., IMGN779) comprise a fully human antibody or antigen-binding fragment thereof.

[0226] In some embodiments, the anti-CD33 antibody or antigen-binding fragment

comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively.

[0227] In some embodiments, the anti-CD33 antibody or antigen-binding fragment

comprises the heavy chain variable domain sequence of SEQ ID NO:9. In some embodiments, the anti-CD33 antibody or antigen-binding fragment comprises the light chain variable domain sequence of SEQ ID NO: 10. In some embodiments, the anti- CD33 antibody or antigen-binding fragment comprises the heavy chain variable domain sequence of SEQ ID NO:9 and the light chain variable domain of SEQ ID NO: 10.

[0228] In some embodiments, the anti-CD33 antibody or antigen-binding fragment

comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively and the heavy chain variable domain sequence of SEQ ID NO:9. In some embodiments, the anti-CD33 antibody or antigen-binding fragment comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively and the light chain variable domain sequence of SEQ ID NO: 10.

[0229] In some embodiments, the anti-CD33 antibody or antigen-binding fragment

thereof comprises the heavy chain sequence of SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 12.

[0230] In a further embodiment, the anti-CD33 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable region and at least one light chain variable region, wherein said heavy chain variable region comprises three

complementarity-determining regions having amino acid sequences represented by SEQ ID NOs: 1-3, respectively, and wherein said light chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs:4-6, respectively.

[0231] In a further embodiment, the anti-CD33 antibody or antigen-binding fragment thereof is a humanized antibody or antigen binding fragment thereof comprising at least one heavy chain variable region and at least one light chain variable region, wherein said heavy chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs: l-3, respectively, and wherein said light chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs:4-6, respectively.

[0232] In a further embodiment, the anti-CD33 antibody or antigen-binding fragment thereof is a CDR-grafted or resurfaced antibody comprising at least one heavy chain variable region and at least one light chain variable region, wherein said heavy chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs: l-3, respectively, and wherein said light chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs:4-6, respectively.

[0233] In a further embodiment, there are provided antibodies or antigen-binding

fragments thereof comprising at least one heavy chain variable region and at least one light chain variable region, wherein said heavy chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs: l, 13, and 3, respectively, and wherein said light chain variable region comprises three complementarity-determining regions having amino acid sequences represented by SEQ ID NOs:4-6, respectively.

[0234] In a further embodiment, antibodies or antigen-binding fragments are provided having a humanized (e.g., resurfaced, CDR-grafted) heavy chain variable region that shares at least 90% sequence identity with an amino acid sequence represented by SEQ ID NO: 9, more preferably 95% sequence identity with SEQ ID NO: 9, most preferably 100% sequence identity with SEQ ID NO:9. In particular embodiments, the antibody includes conservative mutations in the framework region outside of the CDRs.

[0235] Similarly, antibodies are provided having a humanized (e.g., resurfaced, CDR- grafted) light chain variable region that shares at least 90% sequence identity with an amino acid sequence corresponding to SEQ ID NO: 10, more preferably 95% sequence identity with SEQ ID NO: 10, most preferably 100% sequence identity with SEQ ID NO: 10. In particular embodiments, the antibody includes conservative mutations in the framework region outside of the CDRs. [0236] The antibodies and antigen-binding fragments of the immunoconjugate can comprise polypeptides that are recombinant polypeptides, natural polypeptides, or synthetic polypeptides.

[0237] The polypeptides can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0238] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication No. 2008/0312425,

2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein in its entirety.

III. Anti-CD33 Immunoconjugates

[0239] In addition to the anti-CD33 antibody or antigen-binding fragment thereof, the anti-CD33 immunoconjugates (e.g., IMGN779) contain at least one cytotoxin (e.g., DGN462) linked to the antibody or antigen-binding thereof by a linker (e.g., sulfo- SPDB). As used herein, the expression "linked to an anti-CD33 antibody or antigen- binding fragment thereof refers to an immunoconjugate comprising at least one cytotoxin derivative bound to an anti-CD33 antibody or antigen-binding fragment thereof via a suitable linking group, or a precursor thereof.

[0240] The cytotoxin can be a benzodiazepine dimer. The cytotoxin can be an indolino- benzodiazepine dimer. The cytotoxin can be a DNA-alkylating agent. The cytotoxin can be DGN462.

[0241] Immunconjugates can be prepared by using a linking group in order to link the cytotoxin to the anti-CD33 antibody or antigen-binding fragment thereof. Suitable linking groups are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.

[0242] The cytotoxin can, for example, be linked to the anti-CD33 antibody or antigen- binding fragment thereof through a disulfide bond. The linker molecule or crosslinking agent comprises a reactive chemical group that can react with the anti-CD33 antibody or antigen-binding fragment thereof. The reactive chemical groups for reaction with the cell-binding agent can be N-succinimidyl esters and N-sulfosuccinimidyl esters.

Additionally the linker molecule comprises a reactive chemical group, which can be a dithiopyridyl group that can react with the drug to form a disulfide bond. Linker molecules include, for example, N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US Publication No. 20090274713, which is herein incorporated by reference in its entirety). For example, the anti-CD33 antibody or antigen-binding fragment thereof can be modified with crosslinking reagents, and the anti-CD33 antibody or antigen-binding fragment thereof containing free or protected thiol groups thus derived is then reacted with a disulfide- or thiol-containing indolino-benzodiazepine dimer to produce immunoconjugates. The immunoconjugates can be purified by chromatography, including but not limited to HPLC, size-exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration.

[0243] In another aspect of the present invention, the anti-CD33 antibody or antigen- binding fragment thereof is linked to cytotoxic drugs via disulfide bonds and a polyethylene glycol spacer in enhancing the potency, solubility or the efficacy of the immunoconjugate. Such cleavable hydrophilic linkers are described in

WO2009/0134976, which is herein incorporate by reference in its entirety. The additional benefit of this linker design is the desired high monomer ratio and the minimal aggregation of the antibody-drug conjugate. Specifically contemplated in this aspect are conjugates of cell-binding agents and drugs linked via disulfide group (-S-S-) bearing polyethylene glycol spacers ((CH₂CH₂0)_n=₁-₁₄) with a narrow range of drug load of 2-8 are described that show relatively high potent biological activity toward cancer cells and have the desired biochemical properties of high conjugation yield and high monomer ratio with minimal protein aggregation.

[0244] In some embodiments, the linker is a linker containing at least one charged group as described, for example, in U.S. Patent Publication No. 2012/0282282, the contents of which are entirely incorporated herein by reference. In some embodiments, the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the modified cell-binding agent and the cell-binding agent-drug conjugates, especially for monoclonal antibody-drug conjugates with 2 to 20 drugs/antibody linked. Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell. In some embodiments, the linker is N- succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB).

[0245] The immunoconjugate can be an immunoconjugate of Formula (I):

or a pharmaceutically acceptable salt thereof. The double line— between N and C represents either a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen; and when it is a single bond, X is hydrogen and Y is - S0₃H. The term "A" is the anti-CD33 antibody, i.e., an antibody or antigen-binding fragment thereof that specifically binds to CD33 comprising a heavy chain variable region (VH) complementary determining region (CDR) 1 sequence of SEQ ID NO: I, a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and a light chain variable region (VL) CDR1 sequence of SEQ ID NO:4, a VL CDR2 sequence of SEQ ID NO:5, and a VL CDR3 sequence of SEQ ID NO:6. The term "r" is an integer from 1 to 10.

[0246] The immunoconjugates of formulas (IT) and (III), and pharmaceutically acceptable salts thereof, are specific examples of immunoconjugates that can be used in the disclosed methods of treatment.

"A" and "r" are as defined for Formula (I). The term "r" is an integer from 1 to 10. Methods of preparing immunoconjugates of Formulas II and ΠΙ (e.g., IMGN779) are provided in U.S. Patent Nos. 8,765,740 and 9,353,127, the entire teachings of which are incorporated herein by reference.

[0247] In particular embodiments, the antibody portion of the immunoconjugate of formula (I), (Π), or (III) is the huMy9-6 antibody, also termed as "Z4681A." In specific embodiments, the anti-CD33 immunoconjugate is IMGN779. IMGN779 comprises the huMy9-6 or Z4681 A antibody, conjugated to DGN462, via the cleavable disulfide linker N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). IMGN779 is formulated as a combination of Formulas IV and V depicted below:

or a pharmaceutically acceptable salt thereof.

[0248] In certain embodiments, the immunoconjugate comprises 1-10 cytotoxic

benzodiazepine dimer compounds, 2-9 cytototoxic benzodiazepine dimer compounds, 3-8 cytotoxic benzodiazepine dimer compounds, 4-7 cytotoxic benzodiazepine dimer compounds, or 5-6 cytotoxic benzodiazepine dimer compounds.

[0249] In certain embodiments, the immunoconjugate comprises 1-10 indolino- benzodiazepine dimer compounds, 2-9 indolino-benzodiazepine dimer compounds, 3-8 indolino-benzodiazepine dimer compounds, 4-7 indolino-benzodiazepine dimer compounds, or 5-6 indolino-benzodiazepine dimer compounds.

[0250] In certain embodiments, the immunoconjugate comprises 1-10 DGN462, 2-9

DGN462, 3-8 DGN462, 4-7 DGN462, or 5-6 DGN462.

[0251] In certain embodiments, a composition comprising the immunoconjugates

described herein may comprise at least two immunoconjugates with an average 1-10 cytotoxic benzodiazepine dimer molecule per antibody or antigen-binding fragment thereof. The average ratio of cytotoxic benzodiazepine dimer molecule per antibody molecule is referred to herein as the Drug Antibody Ratio (DAR). In one embodiment, the DAR is 2-8, 3-7, 3-5 or 2.5-3.5.

[0252] Further, in some embodiments, the indolino-benzodiazepine dimer (e.g.,

DGN462) is linked (e.g., by sulfo-SPDB) to the anti-CD33 antibody or antigen-binding fragment thereof via a lysine residue of the antibody or antigen-binding fragment thereof. In some embodiments, 1-10 indolino-benzodiazepine dimers (e.g., DGN462) are linked (e.g., by sulfo-SPDB) to the anti-CD33 antibody or antigen-binding fragment thereof via 1-10 lysine residues of the antibody or antigen-binding fragment thereof. In some embodiments, 2-8 indolino-benzodiazepine dimers (e.g., DGN462) are linked (e.g., by sulfo-SPDB) to the anti-CD33 antibody or antigen-binding fragment thereof via 2-8 lysine residues of the antibody or antigen-binding fragment thereof. In some

embodiments, 2-5 indolino-benzodiazepine dimers (e.g., DGN462) are linked (e.g., by sulfo-SPDB) to the anti-CD33 antibody or antigen-binding fragment thereof via 2-5 lysine residues of the antibody or antigen-binding fragment thereof. In some

embodiments, 3-4 indolino-benzodiazepine dimers (e.g., DGN462) are linked (e.g., by sulfo-SPDB) to the anti-CD33 antibody or antigen-binding fragment thereof via 3-4 lysine residues of the antibody or antigen-binding fragment thereof. [0253] The cytotoxic benzodiazepine dimer compound and the conjugates described herein can be prepared according to methods described in U.S. Patent Nos 8,765,740 and 9,353,127, for example, but not limited to, paragraphs [0395]-[0397] and [0598]-[0607], Figures 1, 15, 22, 23, 38-41, 43, 48, 55 and 60, and Examples 1, 6, 12, 13, 20, 21, 22, 23, 26-30 and 32 of U.S. Patent No. 8,765,740 and paragraphs [0007]-[0105], [0197]-[0291], Figures 1-11, 16, 28 and Examples 1-7, 9-13, 15 and 16 of U.S. Patent No. 9,353,127.

[0254] The term "cation" refers to an ion with positive charge. The cation can be

monovalent (e.g., Na⁺, K⁺, etc.), bi-valent (e.g., Ca²⁺, Mg²⁺, etc.) or multi-valent (e.g., Al³⁺ etc.). Preferably, the cation is monovalent.

[0255] The phrase "pharmaceutically acceptable" indicates that the substance or

composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

[0256] The phrase "pharmaceutically acceptable salt" as used herein, refers to

pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate," ethanesulfonate, benzenesulfonate, p- toluenesulfonate, pamoate (i.e., l,l '-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a

pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion. In particular embodiments, the pharmaceutically acceptable salt is a sodium or a potassium salt.

[0257] If the compound of the invention is a base, the desired pharmaceutically

acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

[0258] If the compound of the invention is an acid, the desired pharmaceutically

acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.

Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

IV. Treatment of Cancer Using Anti-CD33 Immunoconjugates

[0259] The present invention provides methods for treating patients with cancer, in

particular a hematologic cancer, such as AML by administering an anti-CD33 immunoconjugate (e.g., IMGN779). In some embodiments, the patient is an unfit AML patient. In some embodiments, the patient is a fit AML patient. As used herein, a "hematologic cancer" is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer are leukemia, lymphoma and multiple myeloma.

[0260] Cancers which can be treated using the disclosed methods include leukemia, lymphoma and myeloma. The cancer can be chemotherapy sensitive; alternatively, the cancer can be chemotherapy resistant. More specifically, cancers which can be treated using the disclosed methods include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute pro-myelocytic leukemia (APL), myelodysplasia syndromes (MDS), acute monocytic leukemia (AMOL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, small lymphocytic lymphoma (SLL), Hodgkin's lymphomas (Nodular sclerosis, Mixed cellularity, Lymphocyte-rich, Lymphocyte depleted or not depleted, and Nodular lymphocyte-predominant Hodgkin lymphoma), non-Hodgkin's lymphomas (all subtypes), chronic lymphocytic leukemia/Small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms (plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, T cell

prolymphocytic leukemia, T cell large granular lymphocytic leukemia, Aggressive NK cell leukemia, Adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma (nasal type), enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma (unspecified), anaplastic large cell lymphoma), and multiple myeloma (plasma cell myeloma Kahler's disease).

[0261] In another embodiment, the cancer is selected from acute myeloid leukemia

(AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome, basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL). In another embodiment, the cancer is acute myeloid leukemia (AML). In yet another embodiment, the acute myeloid leukemia is refractory or relapsed acute myeloid leukemia. In other embodiments, the invention provides treatment of patients with multi-drug resistant AML. P-glycoprotein (PGP), also known as MDR1, is an ATP-dependent drug efflux pump of 170 kD. It is a member of the ABC superfamily and is abundantly expressed in multidrug resistance (MDR) cells and produced by the ABCB1 gene. AML cells expressing PGP are, at least to some degree, resistant to treatment with conventional chemotherapeutics. Thus, the invention also provides methods for treating PGP- expressing AML.

[0262] The invention also provides methods of treating a hematologic cancer having at least one negative prognostic factor, e.g., overexpression of P-glycoprotein,

overexpression of EVI1, a p53 alteration, DNMT3 A mutation, FLT3 internal tandem duplication, and/or complex karyotype. In other embodiments, the invention also provides methods of treating a hematologic cancer having decreased expression in BRCA1, BRCA2, or PALB2 or mutations in BRCA1, BRCA2, or PALB2. Also within the scope of the invention is the selection of patients having at least one negative prognostic factor (e.g., at least one poor cytogenetic factor) and/or decreased expression or mutations in BRCA1, BRCA2, or PALB2 prior to administration of the anti-CD33 immunoconjugate (e.g., IMGN779).

[0263] The invention also provides methods of treating a hematologic cancer having a

RAS mutation. The invention also provides methods of treating a hematologic cancer having a TP53 mutation. The invention also provides methods of treating a hematologic cancer having an IDH mutation. The invention also provides methods of treating a hematologic cancer having an FLT3 mutation.

[0264] Single nucleotide polymorphism (SNPs) have been associated with the efficacy of

CD33-directed therapy. For example, patients with the rsl2459419T polymorphism (discussed for example in Malik et al., Hum. Mol. Genet. 15: 3557-70 (2015)) respond poorly to CD33-directed therapy. Patients with a cytosine (C) instead of a thymine (T) at this SNP location respond more favorably to CD33-directed therapies. Accordingly, in some embodiments, the invention provides methods of treating cancer in patients homozygous for rsl2459419C (CC). In some embodiments, the invention provides methods of treating cancer in patients heterozygous for rsl 2459419C (e.g., CT). Patients undergoing CD33-directed therapy that have the rs35112940A polymorphism (discussed for example in Mortland et al., Clin. Cancer Res. 19: 1620-27 (2013)) exhibit worse overall survival and higher relapse risk in comparison to patients homozygous for the major allele rs35112940G. Accordingly in some embodiments, the invention provides methods of treating cancer in patients homozygous for rs35112940G (GG). In some embodiments, the invention provides methods of treating cancer in patients heterozygous for rs35112940G (e.g., GA). Patients undergoing CD33-directed therapy that have the rsl803254C polymorphism (discussed for example in Mortland et al.) exhibit a higher 3- year treatment-related mortality in comparison to patients homozygous for the major allele rsl803254G. Accordingly in some embodiments, the invention provides methods of treating cancer in patients homozygous for rsl803254G (GG). In some embodiments, the invention provides methods of treating cancer in patients heterozygous for rsl803254G (e.g., GC).

[0265] In some embodiments, the methods comprise administering an anti-CD33

immunoconjugate (e.g., IMGN779) to a patient wherein a homozygous rsl2459419C genotype has been detected in a sample obtained from the patient. In some embodiments, the methods comprise detecting a homozygous rsl 2459419C genotype in sample obtained from the patient and then administering an anti-CD33 immunoconjugate (e.g.,

EVIGN779).

[0266] In some embodiments, the CD33 SNP is detected in a blood sample obtained from the patient. In some embodiments, the CD33 SNP is detected in a buccal swab obtained from the patient.

[0267] In some embodiments, the cancer is a CD33-positive cancer. In some

embodiments, at least 20% of myeloblasts ("blasts") from the cancer are CD33 -positive, e.g., as measured by flow cytometry. In some embodiments, the blasts from the cancer are obtained by bone marrow biopsy.

V. Methods of Administering Anti-CD33 Immunoconjugates

[0268] As provided herein, an anti-CD33 immunoconjugate (e.g., EVIGN779) can be administered at a particular dose. Unless stated otherwise (e.g., in the working

examples), the mg/kg doses are based on antibody. For example, in some embodiments, about 0.02 to about 0.75 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.3 to about 0.75 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.3 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. In some embodiments, about 0.5 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. In some embodiments, about 0.39 to about 0.75 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.39 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. [0269] In some embodiments, about 0.3 to about 0.8 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 0.8 mg kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.5 to about 0.8 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered.

[0270] In some embodiments, about 0.3 to about 0.9 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 0.9 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.5 to about 0.9 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered.

[0271] In some embodiments, about 0.3 to about 1.0 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 1.0 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.5 to about 1.0 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered.

[0272] In some embodiments, about 0.3 to about 1.2 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 1.2 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.5 to about 1.2 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered.

[0273] In some embodiments, about 0.3 to about 1.25 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.4 to about 1.25 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered. In some embodiments, about 0.5 to about 1.25 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered.

[0274] In some embodiments, about 0.54 to about 0.75 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 0.54 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week. In some embodiments, about 0.54 to about 0.8 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 0.54 to about 0.9 mg/kg of an anti- CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 0.54 to about 1.0 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 0.7 to about 1.1 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is

administered, e.g., every other week. In some embodiments, about 0.7 to about 1.15 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 0.7 to about 1.2 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week. In some embodiments, about 0.7 to about 1.25 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week.

[0275] In some embodiments, about 1 to about 1.6 mg/kg of an anti-CD33

immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week. In some embodiments, about 1 to about 2 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered, e.g., every other week.

[0276] In some embodiments, about 1.2 to about 1.6 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered, e.g., every other week. In some embodiments, about 1.2 to about 2 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., every other week.

[0277] In some embodiments, about 1.5 to about 1.6 mg/kg of an anti-CD33

immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week. In some embodiments, about 1.5 to about 2 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered, e.g., every other week.

[0278] In some embodiments, about 0.3 to about 0.54 mg/kg of an anti-CD33

immunoconjugate (e.g., IMGN779) is administered, e.g., weekly. In some embodiments, about 0.39 to about 0.54 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., weekly. In some embodiments, about 0.54 to about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered, e.g., weekly.

[0279] In some embodiments, about 0.39 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered. In some embodiments, about 0.54 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. In some embodiments, about 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, or 0.69 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. In some embodiments, about 0.70 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. In some embodiments, about 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, or 0.80 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered. [0280] In some embodiments, about 0.54 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0281] In some embodiments, about 0.55, 0.56, 0.57, 0.58, or 0.59 mg/kg of an anti-

CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0282] In some embodiments, about 0.6 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0283] In some embodiments, about 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, or 0.69 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0284] In some embodiments, about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0285] In some embodiments, about 0.75 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0286] In some embodiments, about 0.8 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0287] In some embodiments, about 0.85 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0288] In some embodiments, about 0.90 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0289] In some embodiments, about 0.91 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0290] In some embodiments, about 0.92 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. [0291] In some embodiments, about 0.93 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0292] In some embodiments, about 0.94 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0293] In some embodiments, about 0.95 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0294] In some embodiments, about 0.96 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0295] In some embodiments, about 0.97 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0296] In some embodiments, about 0.98 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0297] In some embodiments, about 0.99 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0298] In some embodiments, about 1 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0299] In some embodiments, about 1.01 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.02 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.03 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.04 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.05 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.06 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.07 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.08 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.09 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0300] In some embodiments, about 1.1 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0301] In some embodiments, about 1.11 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.12 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.13 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.14 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.15 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.16 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.17 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.18 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.19 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. [0302] In some embodiments, about 1.2 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0303] In some embodiments, about 1.21 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.22 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.23 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.24 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.25 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0304] In some embodiments, about 1.5 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.51 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.52 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.53 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.54 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.55 mg/kg of an anti-CD33 immunoconjugate (e.g., EVIGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.56 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.57 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.58 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.59 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle. In some embodiments, about 1.6 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0305] In some embodiments, about 1.7 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0306] In some embodiments, about 1.8 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0307] In some embodiments, about 1.9 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0308] In some embodiments, about 1.95 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0309] In some embodiments, about 2 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0310] In some embodiments, about 0.3 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0311] In some embodiments, about 0.35 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0312] In some embodiments, about 0.39 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1 , 8, 15, and 22 of a 28-day cycle.

[0313] In some embodiments, about 0.4 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0314] In some embodiments, about 0.45 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0315] In some embodiments, about 0.50 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0316] In some embodiments, about 0.55 mg/kg of an anti-CD33 immunoconjugate (e.g.,

EVIGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. [0317] In some embodiments, about 0.6 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0318] In some embodiments, about 0.65 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0319] In some embodiments, about 0.7 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0320] In some embodiments, about 0.75 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0321] In some embodiments, about 0.8 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0322] In some embodiments, about 0.85 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0323] In some embodiments, about 0.90 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0324] In some embodiments, about 0.91 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0325] In some embodiments, about 0.92 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0326] In some embodiments, about 0.93 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0327] In some embodiments, about 0.94 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0328] In some embodiments, about 0.95 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0329] In some embodiments, about 0.96 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0330] In some embodiments, about 0.97 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0331] In some embodiments, about 0.98 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0332] In some embodiments, about 0.99 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. [0333] In some embodiments, about 1 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0334] In some embodiments, about 1.01 mg kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.02 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.03 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.04 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.05 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.06 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.07 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1 , 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.08 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.09 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0335] In some embodiments, about 1.1 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0336] In some embodiments, about 1.11 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.12 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.13 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.14 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1 , 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.15 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.16 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.17 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.18 mg/kg of an anti-CD33 immunoconjugate (e.g., IMG 779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.19 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1 , 8, 15, and 22 of a 28-day cycle.

[0337] In some embodiments, about 1.2 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0338] In some embodiments, about 1.21 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.22 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.23 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.24 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.25 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0339] In some embodiments, about 1.5 mg/kg of an anti-CD33 immunoconj ugate (e.g.,

IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.51 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.52 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1 , 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.53 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.54 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.55 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.56 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.57 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.58 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.59 mg/kg of an anti-CD33 immunoconjugate (e.g., IMGN779) is administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, about 1.6 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0340] In some embodiments, about 1.7 mg/kg of an anti-CD33 immunoconjugate (e.g.,

IMGN779) are administered about weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0341] In some embodiments, about 1.8 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0342] In some embodiments, about 1.9 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0343] In some embodiments, about 1.95 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0344] In some embodiments, about 2 mg/kg of an anti-CD33 immunoconjugate (e.g.,

[0345] In some embodiments, an anti-CD33 immunoconjugate (e.g., IMGN779) is

administered weekly, e.g., on days 1, 8, 15, and 22 of a 28-day cycle.

[0346] In some embodiment, an anti-CD33 immunoconjugate (e.g., IMGN779) is

administered about every two weeks, e.g., on days 1 and 15 of a 28-day cycle.

[0347] In some embodiments, an anti-CD33 immunoconjugate (e.g., IMGN779) is

administered to maintain exposure levels or to avoid a significant decrease in exposure.

[0348] In some embodiments, the administration of an anti-CD33 immunoconjugate (e.g.,

IMGN779) results in saturation of residual free-CD33.

[0349] In some embodiments, administration of the administration of an anti-CD33

immunoconjugate (e.g., IMGN779) results in a decrease in peripheral blood blasts. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in a decrease in peripheral blood blasts within 3-8 days of the first dose. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in a decrease in peripheral blood blasts after a second dose. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in at least a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in peripheral blood blasts after a second dose. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) achieves a peripheral blood blasts percentage of about 5%, 4%, 3%, 2%, 1%, or less than 1%.

[0350] In some embodiments, administration of an anti-CD33 immunoconjugate (e.g.,

IMGN779) results in a decrease in bone marrow blasts. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., EVIGN779) results in at least a 40% decrease in bone marrow blasts. In some embodiments, administration of an anti- CD33 immunoconjugate (e.g., EVIGN779) results in at least a 45% decrease in bone marrow blasts. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in at least a 48% decrease in bone marrow blasts. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in at least a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in bone marrow blasts. In some embodiments, administration of an anti-CD33

immunoconjugate (e.g., EVIGN779) results in at least a 59% decrease in bone marrow blasts. In some embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) results in at least a 96% decrease in bone marrow blasts. In some

embodiments, administration of an anti-CD33 immunoconjugate (e.g., IMGN779) achieves a bone marrow blasts percentage of about 5%, 4%, 3%, 2%, 1%, or less than 1%.

[0351] In some embodiments, the anti-CD33 immunoconjugate (e.g., IMGN779) is

administered to a subject in a pharmaceutically acceptable dosage form. Anti-CD33 immunoconjugates (e.g., IMGN779) can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, the anti-CD33 immunoconjugate (e.g., IMGN779) is administered intravenously.

[0352] The anti-CD33 immunoconjugates (e.g. IMGN779) used in the disclosed methods and pharmaceutical compositions can be supplied as a solution or a lyophilized powder that are tested for sterility and for endotoxin levels. Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those of ordinary skill in the art as the clinical situation warrants.

[0353] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1. In vitro potency of IMGN779 in AML cell lines

[0354] EVIGN779 is a specific, targeted antibody drug conjugate (ADC) that binds with high affinity to CD33, which is widely expressed on acute myeloid leukemia (AML) cells but not in non-hematopoietic tissues. EVIGN779 has been previously described in International Published Application Nos. WO2015/179400 and WO2012/128868, and U.S. Published Application Nos. US2017/0080102 and US2015/009987, each of which is incorporated by reference herein in its entirety. IMGN779 comprises a humanized anti CD33 antibody (Z4681A) attached via a disulfide containing linker to the DNA- alkylating payload DGN462 (approximately 3 payloads per antibody). Z4681A antibody contains a variable heavy chain with the amino acid sequence of SEQ ID NO: 9 and a variable light chain with the amino acid sequence of SEQ ID NO: 10. The Z4681 A variable heavy chain comprises CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: l-3, and the Z4681A variable light chain comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:4-6. Once released within the target cell, DGN462 exerts its anti-tumor effect by alkylating DNA, without cross-linking, resulting in cell cycle arrest and apoptosis.

[0355] Potency of IMGN779 was tested on two AML cell lines, EOL- 1 and MV4- 11 , in in vitro cytotoxicity assays.

[0356] Cytotoxic assays were performed in triplicate for each data point. To block Fc receptors on AML cells, the culture medium was supplemented with 100 nM of huKTI. AML cells were added to each well of a flat bottom 96-well plates. The test articles were diluted in complete cell culture medium and the dilutions were added to the plated cells. Wells containing cells and the medium but lacking the test articles, as well as wells containing medium only, were included in each assay plate. The plates were incubated at 37°C in a humidified 6% C02 incubator for four days. The relative number of viable cells in each well was then determined using the Water Soluble Tetrazolium Salt-8 (WST-8) based Cell Counting Kit. The WST-8 reagent was added to a final volume of 10% and plates were incubated at 37°C for an additional two to eight hours. The absorbance of each well was measured on a plate-reader spectrophotometer in the dual wavelength mode 450nm/650nm, and the absorbance at 650nm (non-specific light scattering by cells) was subtracted from the 450nm absorbance. The surviving fraction of cells in each well was calculated by subtracting the average value of the wells containing medium only, and dividing by the average value of the wells containing cells and the medium but lacking the test articles. The surviving fractions were plotted against the test article concentrations in a semi-log plot. IC50 values were estimated from the plotted survival curves.

[0357] As shown in FIGs. 1A and IB, IMGN779 was cytotoxic to both cell lines tested, killing EOL-1 cells with an IC50 of lOnM and MV4-11 cells with an IC50 of 5nM (solid lines in FIGs. 1A and IB). Moreover, the killing was CD33 -dependent since blocking CD33 with unconjugated antibody (dotted lines in FIGs. 1A and IB) decreased

EVIGN779 cytotoxicity at least 10 fold.

Example 2. In vitro cytotoxicity and apoptotic activity of DNA crosslinking vs. DNA alkylating payloads in AML cell lines

[0358] The potency of the DNA crosslinking payload, DGN484 and the DNA alkylating payload, DGN462 in EVIGN779 were tested on three AML cell lines, EOL-1, HL-60, and OCI-M1, in in vitro cytotoxicity assays. DGN484 is disclosed in U.S. Publication No. 2016-0108129 as compound 1H.

[0359] Cytotoxic assays were performed in triplicate for each data point. Cell lines were cultivated as previously described. Cells in culture were plated into 96 well plates at 50,000 live cells/well. Serial dilutions of either DGN462 or DGN484 were added to the cells in 96 well plates, for total volumes of 200 μL/well. Wells with media only served as negative control and cells in media treated with DGN484/DGN462 solvent at a concentration that matched the greatest serial dilution molarity served as a positive control. After 72 hours of culture, 20 μL of WST-8 was added per 200 μL in each well. Plates were replaced into incubators. After 2-8 hours of WST-8 exposure, spectrophotometry was used to assess the relative quantities of live cells (SpectraMax M2; at 450 nm).

[0360] As seen in FIG. 2A, EOL-1 cells are more sensitive to DGN462 and DGN484 than HL60 and OCI-M1 cells. Moreover, the crosslinking agent, DGN484 is more potent than the alkylating agent, DGN462 in vitro in all cell lines tested.

[0361] The apoptotic activity of the crosslinking and alkylating agents were also tested in these AML cell lines. In these experiments, cell lines were cultivated as previously described. Cells in culture were harvested and seeded in cell culture flasks at 500,000 live cells/mL. Either DGN462, DGN484, or the DGN462/DGN484 vehicle (negative control) was added to the flasks. After both 24 and 48 hours of growth in culture, 2 mL per culture was harvested for staining with fluorochrome-conjugated Annexin V and To- Pro®-3 Iodide. Cells were then run on the BD FACSCanto™ Π and results were processed in FACSDiva 10 and graphed.

[0362] As seen in FIG. 2B, treatment with DGN484 in vitro resulted in more apoptotic cells than did treatment with an equal dose of DGN462 in the cell lines tested. The magnitude of this difference was greater in cell lines with greater DGN sensitivity.

Example 3. In vitro cytotoxicity of DNA crosslinking vs. DNA alkylating conjugatess in AML cell lines

[0363] The potency of EVIGN779 conjugate and the CD33- targeting cross-linker

conjugate (i.e., the Z4681 A antibody conjugated to the DNA crosslinking payload, DGN484 via a sSPDB linker) were tested on AML cell lines in in vitro cytotoxicity assays.

[0364] Assays were performed in triplicate for each data point. To block Fc receptors on

AML cells, the culture medium was supplemented with 100 nM of huKTI. AML cells, 1,500 to 10,000 in 100 μL of complete culture medium, were added to each well of flat bottom 96-well plates. The test articles were diluted in complete cell culture medium and 100 μL of the dilutions were added to the plated cells. Wells containing cells and the medium but lacking the test articles, as well as wells containing medium only, were included in each assay plate. The plates were incubated at 37 °C in a humidified 6% C0₂ incubator for four to seven days. Then, the relative number of viable cells in each well was determined using the Water Soluble Tetrazolium Salt-8 (WST-8) based Cell

Counting Kit (Dojindo Molecular Technologies, Inc., Rockville, MD). The WST-8 reagent was added to a final volume of 10% and plates were incubated at 37 °C for an additional two to six hours. The absorbance of each well was measured on a plate-reader spectrophotometer in the dual wavelength mode 450nm/650nm, and then the absorbance at 650nm (non-specific light scattering by cells) was subtracted from the 450nm absorbance. The surviving fraction of cells in each well was calculated by subtracting the average value of the wells containing medium only, and then dividing by the average value of the wells containing cells and the medium but lacking the test articles. The surviving fractions were plotted against the test article concentrations in a semi-log plot. IC₅₀ values were estimated from the plotted survival curves.

[0365] The binding of IMGN779 and the CD33 -targeting cross-linker conjugate to

MOLM-13 cells are similar as assessed by an indirect binding assay (see, FIG. 3). As seen in FIGs. 4A-4D, the CD33 -targeting conjugate of DGN484 is 1.6 to 5-fold more active than IMGN779 against AML cell lines in vitro.

Example 4. Tolerability ofIMGN779, Z4681A-S-SPDB-DGN484 and Related

Conjugates in Mice, Rats, and Cynomolgus Monkeys

A. Intravenous Single Dose Tolerability of IMGN779 in Female CD-I Mice

[0366] To test the tolerability of IMGN779 in vivo, a mouse model was used as described in the protocol below.

[0367] Female CD-I mice received a single intravenous injection into the lateral tail vein of vehicle, 650 μg/kg (by DGN462, 32.9 mg/kg by antibody) of IMGN779, 725 μg/kg (by DGN462, 36.7 mg/kg by antibody) of BVIGN779, 800 μg/kg (by DGN462, 40.5 mg/kg by antibody) of IMGN779, 875 μg/kg (by DGN462, 44.3 mg/kg by antibody) of IMGN779, 950 μg/kg (by DGN462, 48.1 mg/kg by antibody) of IMGN779, or 1025 μg/kg (by DGN462, 51.9 mg/kg by antibody) of IMGN779. The antibody does not cross- react with mouse CD33, which makes this in vivo mouse model an indicator of off-target toxicity only. The mice were observed daily for 19 days, and body weights were determined. If an animal experienced greater than 20% body weight loss or became moribund, the animal was euthanized. Percent BW Change is calculated from the following formula: %BW Change = [(BWpost / BWpre)-l] xlOO; where BWpost is weight after treatment and BWpre is the starting body weight prior to treatment. A positive % BW change indicates BW gain, while a negative % BW change indicates the loss of BW. [0368] The results are summarized in Table 3 (below) and in FIG. 5. IMGN779 was tolerated at 650 μ§/1¾ (by DGN462, 32.9 mg/kg by antibody). The nadir of mean change in body weight occurred on day 3, with a 4% decrease. None of the eight mice in this treatment group were euthanized due to body weight loss. IMGN779 was tolerated at 725 μg/kg (by DGN462, 36.7 mg/kg by antibody). The nadir of mean change in body weight occurred on day 5, with a 6% decrease. None of the eight mice in this treatment group were euthanized due to body weight loss. IMGN779 was tolerated at 800 μg/kg (by DGN462, 40.5 mg/kg by antibody). The nadir of mean change in body weight occurred on day 4, with an 8% decrease. None of the eight mice in this treatment group were euthanized due to body weight loss. IMGN779 was tolerated at 875 μg/kg (by DGN462, 44.3 mg/kg by antibody). The nadir of mean change in body weight occurred on day 6, with a 6% decrease. None of the eight mice in this treatment group were euthanized due to body weight loss. IMGN779 was tolerated at 950 μg/kg (by DGN462, 48.1 mg/kg by antibody). The nadir of mean change in body weight occurred on day 5, with a 9% decrease. None of the eight mice in this treatment group were euthanized due to body weight loss. This dose defines to maximum tolerated dose (MTD) for IMGN779 in mice. IMGN779 was not tolerated at 1025 μg/kg (by DGN462, 51.9 mg/kg by antibody). The nadir of mean change in body weight occurred on day 6, with an 11% decrease. Two out of eight of the mice in this treatment group were euthanized due to body weight loss.

TABLE 3:

B. Intravenous Single Dose Tolerability ofIMGN779 or Z4681A-S-SPDB-DGN484 in Female CD-I Mice

[0369] To test the tolerability of IMGN779 or Z4681 A-S-SPDB-DGN484 in vivo, a

mouse model was used as described in the protocol below.

[0370] Female CD-I mice received a single intravenous injection into the lateral tail vein of vehicle, 475 (by DGN462, 24.7 mg/kg by antibody) of IMGN779, 475 μg/kg (by DGN484, 25.7 mg/kg by antibody) of Z4681A-S-SPDB-DGN484, 360 μg/kg (by DGN484, 19.4 mg/kg by antibody) of Z4681A-S-SPDB-DGN484, 250 μg/kg (by

DGN484, 13.5 mg/kg by antibody) of Z4681A-S-SPDB-DGN484, or 190 μg/kg (by DGN484, 10.3 mg/kg by antibody) of Z4681A-S-SPDB-DGN484. The antibody does not cross-react with mouse CD33, which makes this in vivo mouse model an indicator of off- target toxicity only. The mice were observed daily for 90 days, and body weights were determined. If an animal experienced greater than 20% body weight loss or became moribund, the animal was euthanized. Percent BW Change is calculated from the following formula: %BW Change = [(BWpost / BWpre)-l] xlOO; where BWpost is weight after treatment and BWpre is the starting body weight prior to treatment. A positive % BW change indicates BW gain, while a negative % BW change indicates the loss of BW.

[0371] The results are summarized in Table 4 (below) and in FIG. 6. IMGN779 was tolerated at a dose of 475 ug/kg (by DGN462, 24.7 mg/kg by antibody), with no animals euthanized prior to the end of the study (day 90). Other than minimal, individual animal BW loss, no other clinical observations were made during the course of the study. The group mean (N = 8) BW loss nadir occurred on day 4 (-4.2%).

[0372] Z4681A-S-SPDB-DGN484 at a dose of 190 μg/kg (by DGN484, 10.3 mg/kg by antibody) was not tolerated, with four out of the eight animals euthanized due to >20% BW loss (one each on day 24, day 33, day 47 and day 57) and two animals euthanized due to >20% BW loss accompanied by hind limb paralysis (both on day 33) prior to the end of the study. Two out of eight mice survived to the end of the study (day 90). The group mean (N = 8) BW loss nadir occurred on day 24 (-5.2%). Z4681A-S-SPDB- DGN484 at a dose of 250 μg/kg (by DGN484, 13.5 mg/kg by antibody) was not tolerated, with two out of the eight animals euthanized due to >20% BW loss (one each on day 20 and day 23), three animals euthanized due to hind limp paralysis (one each on day 28, day 29 and day 30), two animals euthanized due to body weight loss accompanied by hind limb paralysis (day 26, day 28) and one animal euthanized due to scoliosis (day 20) prior to the end of the study. The group mean (N = 8) BW loss nadir occurred on day 20 (- 10.4%). Z4681A-S-SPDB-DGN484 at a dose of 360 g/kg (by DGN484, 19.4 mg/kg by antibody) was not tolerated, with five out of the eight mice euthanized due to >20% BW loss (two on day 17, and one each on day 23, day 24 and day 26) and three mice euthanized for >20% BW loss accompanied by hind limb paralysis (one each on day 16, day 19 and day 28) prior to the end of the study. The group mean (N = 8) BW loss nadir occurred on day 16 (-13.5%). Z4681A-S-SPDB-DGN484 at a dose of 475 ug/kg (by DGN484, 25.7 mg/kg by antibody) was not tolerated, with four out of the eight animals euthanized due to >20% BW loss (one each on day 17, day 18, day 23 and day 26), three animals euthanized for morbidity (one each on day 13, day 19 and day 20) and one animal euthanized for morbidity accompanied by hind limb paralysis (day 18) prior to the end of the study. The group mean (N = 8) BW loss nadir occurred on day 4 (-5.3).

[0373] A serious clinical observation noted for mice treated with each of the four

different dose levels of Z4781A-S-SPDB-DGN484 (190, 250, 360 or 475 μg/kg based on DGN484 concentration) was hind limb paralysis, which suggests that treatment with this conjugate is ultimately having some kind of a neurological impact in these non-tumor bearing CD-I mice. Hind limb paralysis was not observed in any of the eight mice treated with 475 ug/kg (based on DGN462 concentration) of IMGN779.

TABLE 4:

C. Toxicity of IMGN779 in Mice, Rats, and Cynomolgus Monkeys

[0374] In three different toxicology studies initiated in three different species (at a

contract research organization), IMGN779 was administered as a single intravenous injection to the animals at 36 mg/kg (by antibody) to female CD-I mice, at 5 mg/kg (by antibody) to female Sprague Dawley rats, and at 2.4 mg/kg (by antibody) to female cynomolgus monkeys. Liver enzymes and blood cellularity were measured by standard methods. Body weight was measured over the course of each study, and clinical observations were recorded. At the end of each study, animals were euthanized and organs and tissues were collected, fixed and processed into hematoxylin and eosin (H&E)-stained slides, which were examined by veterinary pathologists, who recorded their findings. The results of these three toxicology studies are summarized in Table 5 (below).

[0375] In mice, IMGN779, administered at 36 mg/kg (by antibody) as a single

intravenous injection, caused mild elevation of liver enzymes (ALT, AST, SDH). The major histopathological findings noted were gastrointestinal (GI) damage and bone marrow depletion. The major clinical observation was body weight loss. When peripheral blood cellularity was analyzed, decreases were found in thrombocytes (mild to moderate), reticulocytes (severe), neutrophils (moderate) and lymphocytes (moderate).

[0376] In rats, IMGN779, administered at 5 mg/kg (by antibody) as a single intravenous injection, caused slight elevation of SDH and a slight decrease in ALT and ALP. The major histopathological findings noted were gastrointestinal (GI) damage and bone marrow depletion. The major clinical observation was body weight loss and diarrhea at doses exceeding the maximum tolerated dose (MTD). When peripheral blood cellularity was analyzed, decreases were found in thrombocytes (mild to moderate), reticulocytes (severe), neutrophils (moderate to severe), and lymphocytes (moderate).

[0377] In cynomolgus monkeys, IMGN779, administered at 2.4 mg/kg (by antibody) as a single intravenous injection, caused no changes in the levels of liver enzymes (ALT, AST, SDH). The major histopathological findings noted were gastrointestinal (GI) damage and bone marrow depletion. The major clinical observation was body weight loss and diarrhea. When peripheral blood cellularity was analyzed, decreases were found in thrombocytes (mild to moderate), reticulocytes (severe), neutrophils (moderate to severe), and lymphocytes (mild).

TABLE 5:

D. Toxicity of IMGN779 (cleavable linker) and a CD33-Targeting Conjugate

without a Cleavable Linker (Z4681A-IGN23) in Female CD-I Mice

[0378] IMGN779, which has a cleavable -s-SPDB- linker, or Z4681 A-IGN23, a mono- imine payload-bearing conjugate with a non-cleavable linker, was administered to female CD-I mice as a single intravenous injection. IGN23 is disclosed in U.S. Publication No. 2016-0222013 as compound 254C. IMGN779 was administered at doses of

approximately 12.5 mg/kg (by antibody) and approximately 35 mg/kg (by antibody), and Z4681A-IGN23 was administered at a dose of approximately 10 mg/kg (by antibody). Blood samples were taken five days post-conjugate administration and were analyzed for levels of liver enzymes (ALT, AST, SDH) and for cellularity (white blood cells, platelets, reticulocytes, neutrophils and lymphocytes) using standard methods. The percent change (% change) for each parameter was calculated as follows: % change = ((value, conjugate- treated mice - value, untreated mice)/ value, untreated mice) x 100%, where historical values for normal, untreated, female CD-I mice were used as the untreated baseline levels for each parameter. A positive % change represents an increase compared to baseline levels, while a negative % change represents a decrease compared to baseline levels, where a 100% decrease would indicate that the parameter's value was reduced to zero. The results are summarized in Table 6 (below).

[0379] On day 5 post-conjugate administration, treatment with the lower dose of 12.5 mg/kg of EVIGN779 lead to mild elevations in liver enzymes, with a 45% increase in ALT, a 39% increase in AST and a 46% increase in SDH. Similarly, treatment with the 35 mg/kg dose of IMGN779 increased liver enzymes, but to a greater extent than the 12.5 mg/kg dose, with a 393% increase in ALT, a 135% increase in AST and a 275% increase in SDH. In contrast, treatment with 10 mg/kg dose of Z4681 A-IGN23 resulted in very large increases in liver enzymes, with a 14,000% increase in ALT, a 6,000% increase in AST and a 750% increase in SDH above baseline levels.

[0380] On day 5 post-conjugate administration, treatment with the lower dose of 12.5 mg/kg of IMGN779 lead to decreases in white blood cells (WBCs, 67%), reticulocytes (98%), neutrophils (83%) and lymphocytes (63%), and a very small increase in platelets (9.4%). In contrast, treatment with Z4681 A-IGN23 at a dose of 10 mg/kg (by antibody), which was equal to 80% of the lower dose of IMGN779, resulted in a 91% decrease in platelets. In addition, treatment with the 10 mg/kg dose of Z4681 A-IGN23 resulted in a 10% decrease in WBCs, a 73% decrease in reticulocytes, a 200% increase in neutrophils and a 47% decrease in lymphocytes. Treatment with the high dose of IMGN779, 35 mg/kg by antibody, lead to a 79% decrease in WBCs, a 77% decrease in platelets, a 99% decrease in reticulocytes, a 92% decrease in neutrophils and a 76% decrease in lymphocytes.

TABLE 6:

Example 5. In Vivo Efficacy of Single-Dose IMGN779 in EOL-1 Subcutaneous Model Data collection and analysis for all subcutaneous xenograft models

[0381] Mice were weighed twice a week and were monitored for clinical signs

throughout the duration of the study. Animals were euthanized when hind leg paralysis was present, body weight decreased by >20% of pre-treatment weight, tumor ulceration occurred, or when any signs of distress were visible. Tumor volumes were measured one to two times weekly in three dimensions using a caliper. The tumor volume was expressed in mni3 using the formula V = Length x Width x Height x ½ (Tomayko and Reynolds, Cancer Chemother. Pharmacol. 24: 148-54 (1989)). Activity was assessed as described in Bissery et al., Cancer Res. 51 : 4845-52 (1991). Tumor Growth Inhibition (T/C Value) was also assessed using the following formula: T/C (%) = (Median tumor volume of the treated / Median tumor volume of the control) x 100%. Tumor volume was determined simultaneously for the treated (T) and the vehicle control (C) groups when tumor volume of the vehicle control reached a predetermined size (Bissery et al., Cancer Res. 51 : 4845-52 (1991). The daily median tumor volume of each treated group was determined, including tumor-free mice (0 mni3). According to NCI standards, a T/C < 42% is the minimum level of anti -tumor activity. A T/C < 10% is considered a high antitumor activity level.

[0382] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a subcutaneous tumor model was used as described in the protocol below.

[0383] Female athymic nude mice were each inoculated with 1x107 EOL-1 cells, a

human AML cell line, in 100 ul serum free medium/matrigel subcutaneously in the right flank. On day 6 post-EOL-1 inoculation, mice were randomized into the study groups based on tumor volume. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted, humanized AVE1642 IgG antibody to block Fc receptors on the EOL-1 cells, preventing non-specific up-take of conjugate. Similarly, on days 5 and 10 post-conjugate administration (days 10 and 15 post-EOL-1 inoculation), the mice were injected intraperitoneally with 100 mg/kg of AVE1642 antibody to ensure continued blocking of Fc receptors on the EOL-1 cells. On day 6 post- EOL-1 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.602 mg/kg by antibody) IMGN779, 30 μg/kg (by DGN462; 1.80 mg/kg by antibody) IMGN779, 60 g/kg (by DGN462; 3.61 mg/kg by antibody) IMGN779, 10 μg/kg (by DGN462; 0.424 mg/kg by antibody) chKTI-DGN462, 30 μg/kg (by DGN462; 1.27 mg/kg by antibody) chKTI-DGN462 or 60 μg/kg (by DGN462; 2.54 mg/kg by antibody) chKTI-DGN462.

[0384] The results are represented in Table 7 (below) and in FIG. 7. BVIGN779 was highly active at all dose levels tested in this study. At both 60 μg/kg and 30 μg/kg of EVIGN779, the T/C values were 0%, with 6/6 complete regressions (CRs), and all animals remained tumor free until the study was terminated on day 90, although one animal had to be sacrificed in the highest dose group at day 67 due to morbidity. At the lowest dose of 10 μg/kg, IMGN779 had a T/C value of 3%, 6/6 partial regressions (PR) and 6/6 CRs. The non-targeting chKTI-DGN462 conjugate was inactive at the 10 μg/kg dose, with a T/C of 78% and no PRs or CRs, and was active at 30 μg/kg, with a T/C of 20% but with only 1/6 PRs and 1/6 CRs. At the highest dose of 60 μg/kg, the chKTI-DGN462 non- targeted conjugate was highly active, with a T/C value of 3%, with 5/6 PRs and 5/6 CRs.

TABLE 7:

Example 6. In Vivo Efficacy of Single Dose IMGN779 in HL60/QC Subcutaneous Model

[0385] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a subcutaneous tumor model was used as described in the protocol below.

[0386] Female C.B17 SCID mice were each inoculated with 5x106 HL60/QC cells, a human AML cell line, in 100 ul serum free medium/matrigel subcutaneously in the right flank. On day 9 post-HL60/QC inoculation, mice were randomized into the study groups. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the HL60/QC cells, preventing non-specific up-take of conjugate. Similarly, on days 4 and 9 post-conjugate administration, the mice were injected intraperitoneally with 100 mg/kg of non-targeted chKTI Ab to ensure continued blocking of Fc receptors on the HL60/QC cells. On day 10 post-HL60/QC inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.520 mg/kg by antibody) IMGN779, 5 μg/kg (by DGN462; 0.260 mg/kg by antibody) IMGN779 or 2.5 μg/kg (by DGN462; 0.130 mg/kg by antibody) IMGN779.

[0387] The results are represented in Table 8 (below) and in FIG. 8. The 10 μg/kg dose

(by DGN462) of EVIGN779 was highly active, generating a 7% T/C and 1/6 complete regressions (CRs). The 5 and 2 μg/kg doses (by DGN462) of EVIGN779 were both inactive, generating a 46% T/C (0/6 CRs) and a 76% T/C (0/6 CRs), respectively. These results define the minimum effective dose for a single intravenous administration of EVIGN779 as 10 ug/kg in the HL60/QC subcutaneous model.

TABLE 8:

Example 7. In Vivo Efficacy of Multiple Doses ofIMGN779 in HL60/QC

Subcutaneous Model

[0388] To test the efficacy of IMGN779 for the ability to decrease tumor burden

a subcutaneous tumor model was used as described in the protocol below. [0389] Female C.B17 SCID mice were each inoculated with 5x106 HL60/QC cells, a human AML cell line, in 100 μΐ serum free medium/matrigel subcutaneously in the right flank. Unlike in previous studies, groups were not treated with excess human IgG for FcR blocking to eliminate nonspecific activity, as FcR was shown not to contribute to activity at the low doses tested in this study. On day 9 post-cell inoculation, the mice were randomized into study groups based on tumor volume. On day 9 post-HL60/QC inoculation (day 1 of treatment), the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.506 mg/kg by antibody) IMGN779, 30 μg/kg (by DGN462; 1.52 mg/kg by antibody) IMGN779 or 0.66 mg/kg of

unconjugated Z4681A antibody plus 30 μg/kg of free DGN462 payload. Certain groups in the study received additional single intravenous injections, in the lateral tail vein, of 10 μg/kg (by DGN462; 0.506 mg/kg by antibody) IMGN779 either on day 4 and 7

(completing a Q3D x 3 dosing regimen) or on days 8 and 15 (completing a QW x 3 dosing regimen) following the first IMGN779 treatment (day 1). Certain other groups in the study received additional single intravenous injections, in the lateral tail vein, of 30 μg/kg (by DGN462; 1.52 mg/kg by antibody) IMGN779 either on days 4 and 7

(completing a Q3D x 3 dosing regimen) or on days 8 and 15 (completing a QW x 3 dosing regimen) following the first IMGN779 treatment (day 1).

[0390] The results are represented in Table 9 (below) and in FIG. 9. IMGN779 was active at 10 μg/kg (by DGN462) at both the single and the QW x 3 doses (days 1, 8 and 15), with T/C values of 12% and 10%, respectively, and was highly active when dosed at 10 ug/kg (by DGN462) Q3D x 3 (days 1, 4 and 7), with a T/C value of 7%. In the single dose group there were 3/6 partial regressions (PR) and 3/6 complete regressions (CR), with no animals remaining tumors free at the end of the study (Day 90). In both the QW x 3 and the Q3D x 3 groups, there were 6/6 PRs and 6/6 CRs, and 2/6 animals in each group were tumor free at the end of the study (day 90). At the higher dose of 30 μg/kg (by DGN462), IMGN779 was highly active at all dose schedules, with T/C values of 7% for the single dose and 6% for the Q3D x 3 and the QW x 3 groups. All three groups had 6/6 PRs and 6/6 CRs. The single dose group and the QW x 3 group had 5/6 tumor free survivors and the Q3D x 3 group had 4/6 tumor-free survivors at the end of the study (day 90). A single administration of Z4681A antibody, at 0.66 mg/kg, and free DGN462 payload, at 30 μg/kg, was inactive, with a 92 % T/C and 0/6 PRs and CRs. TABLE 9:

Example 8. In Vivo Efficacy of Single Doses ofIMGN779 or Z4681A-S-SPDB- DGN484 in HL60/QC Subcutaneous Model

[0391] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a subcutaneous tumor model was used as described in the protocol below.

[0392] Female C.B 17 SCID mice were each inoculated with 5x 106 HL60/QC cells, a human AML cell line, in 100 μΐ serum free medium/matrigel subcutaneously in the right flank. On day 9 post-HL60/QC inoculation, mice were randomized into the study groups. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the HL60/QC cells, preventing non-specific up-take of conjugate. Similarly, on days 4 and 9 post-conjugate administration, the mice were injected intraperitoneally with 100 mg/kg of non-targeted chKTI Ab to ensure continued blocking of Fc receptors on the HL60/QC cells. On day 10 post-HL60/QC inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 30 μg/kg (by DGN462; 1.56 mg/kg by antibody) IMGN779, 10 μg/kg (by DGN462; 0.522 mg/kg by antibody) IMGN779, 10 μg/kg (by DGN484; 0.473 mg/kg by antibody) chKTI-s-SPDB-DGN484, 10 μg/kg (by DGN484, 0.541 mg/kg by antibody) Z4681A-S-SPDB-DGN484, 5 μg/kg (by DGN484, 0.271 mg/kg by antibody) Z4681A-S-SPDB-DGN484, 2.5 μg/kg (by DGN484, 0.135 mg/kg by antibody) Z4681A-S-SPDB-DGN484, 1 (by DGN484, 0.0541 mg/kg by antibody) Z4681A-S- SPDB-DGN484 or 0.5 μg/kg (by DGN484, 0.0271 mg/kg by antibody) Z4681A-S-SPDB- DGN484.

[0393] The results are represented in Table 10 (below) and in FIG. 10. The 30 μg/kg dose (by DGN462) of IMGN779 was highly active, generating a 4% T/C, 6/6 PRs and 6/6 CRs. The 10 μg/kg dose of IMGN779 was active, generating a 15% T/C, 4/6 PRs and 3/6 CRs. The 10 ug/kg dose (by DGN484) of Z4681A-S-SPDB-DGN484 was highly active, generating a 5% T/C, 6/6 PRs and 4/6 CRs. The 5, 2.5, 1 and 0.5 μg/kg doses of Z4681A- S-SPDB-DGN484 were all inactive, generating a %T/C values of 43, 88, 97 and 99, respectively, with no PRs or CRs achieved in any of these treatment groups. The non- targeted chKTI-s-SPDB-DGN484 control conjugate dosed at 10 μg/kg (by DGN484) was inactive and generated a 104% T/C value, with no PRs or CRs.

TABLE 10:

Example 9. In Vivo Efficacy of A Single Dose vs. Multiple Fractionated Doses of IMGN779 in MV4-11 Subcutaneous Model

[0394] To test the efficacy of EVIGN779 for the ability to decrease tumor burden in vivo, a subcutaneous tumor model was used as described in the protocol below.

[0395] Female C.B17 SCID mice were each inoculated with 10x106 MV4-11 cells, a human AML cell line, in 100 μΐ serum free medium/matrigel subcutaneously in the right flank. On day 13 post-HL60/QC inoculation, mice were randomized into the study groups based on tumor volume. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 cells, preventing non-specific up-take of conjugate. Similarly, on days 18 and 23 post-MV4-l 1 inoculation, the mice were injected intraperitoneally with 100 mg/kg of non-targeted chKTI Ab to ensure continued blocking of Fc receptors on the MV4-1 1 cells. On day 14 post-MV4-l 1 inoculation (day 1 of treatment), the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.506 mg/kg by antibody) IMGN779 or 5 μg/kg (by DGN462; 0.253 mg/kg by antibody) EVIGN779. Following this initial dose, one of the 5 μg/kg EVIGN779-dosed groups received a second single intravenous injection of 5 μg/kg (by DGN462) of IMGN779 on day 4 treatment (completing a Q3D x 2 dosing regimen) and the other 5 μg/kg EVIGN779-dosed group received a second single intravenous injection of 5 μg/kg (by DGN462) of IMGN779 on day 8 of treatment (completing a QW x 2 dosing regimen)

The results are represented in Table 11 (below) and in FIG. 11. IMGN779 was highly active at the single dose of 10 μg/kg (by DGN462) and at both divided dose schedules (5 μg/kg, Q3D x 2 and QW x 2), with T/C values of 0 % in all groups. There were complete regressions (CR) in 6/6 animals in the 10 ug/kg single dose group, in 5/6 animals in the 5 ug/kg Q3D x 2 group and in 6/6 animals in the 5 μg kg QW x 2 divided dose group. Each of the two fractionated dose groups had more tumor-free survivors (5/6 in both groups) at the end of the study (day 90) compared to the single dose group with 3/6 tumor-free survivors.

TABLE 11 :

Example 10. In Vivo Efficacy of Single Dose IMGN779 in MV4-11 Disseminated ModelData collection and analysis for all disseminated xenograft models

[0397] The mice were weighed twice a week and were monitored for clinical signs

throughout the duration of the study. The measured end-point was survival. Animals were euthanized when hind leg paralysis was present, body weight decreased by >20% of pre-treatment weight, a visible tumor appeared, or any signs of distress were visible. Spontaneous deaths were recorded when they were discovered.

[0398] For disseminated models, Tumor Growth Delay is calculated as T-C, where T is the median survival time (in days) of a treated group and C is the median survival time (in days) of the vehicle control group. The Percent Increased Life Span (%ILS) for disseminated models is calculated using the following formula: %ILS = (T-C) / C x 100%. Anti -tumor activity was evaluated as per NCI standards for disseminated models: ILS > 25% is minimum active, ILS > 40% is active, and ILS > 50% is highly active.

[0399] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a disseminated tumor model was used as described in the protocol below.

[0400] Female NOD SCID mice were pre-treated with 150 mg/kg cyclophosphamide to partially ablate bone marrow in order to improve the engraftment of MV4-11 cells. The cyclophosphamide (Baxter, Lot # 4E011, Exp. 05/2017) was reconstituted with 0.9% NaCl and was administered intraperitoneally to the mice on day -3 and day -2 prior to MV4-11 cell inoculation on day 0. Following cyclophosphamide treatment as described above, the mice were each injected intravenously in the lateral tail vein with 3x106 MV4- 11 cells, a human AML cell line, in 100 μΐ of serum-free medium. On day 6 post-MV4- 11 inoculation, mice were randomized into the study groups based on body weight. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 AML cells, preventing non-specific up-take of conjugate. On day 7 post-MV4-l 1 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.528 mg/kg by antibody) IMGN779, 60 μg/kg (by DGN462; 3.17 mg/kg by antibody) IMGN779, 10 μg/kg (by DGN462; 0.560 mg/kg by antibody) chKTI-s- SPDB-DGN462 or 60 μg/kg (by DGN462; 3.36 mg/kg by antibody) chKTI-s-SPDB- DGN462.

[0401] The results are represented in Table 12 (below) and in FIG. 12. IMGN779, at 60 μg/kg, was highly active in this model, with an Increased Life Span (ILS) of 145.3% and a Tumor Growth Delay (T-C) of 78.5 days, with three animals surviving at study termination. IMGN779, at 10 μg/kg, was also highly active, with an ILS of 90.7% and a Tumor Growth Delay of 49 days, with one animal surviving at study termination. The non-targeted conjugate, chKTI-s-SPDB-DGN462 was active at 60 μg/kg with an ILS of 42.6% and a Tumor Growth Delay of 23 days, with no surviving animals at study termination. At 10 μg/kg, chKTI-s-SPDB-DGN462 was inactive, with an ILS of 4. and a Tumor Growth Delay of 2.5 days, with no surviving animals at study end.

TABLE 12:

Example 11. In Vivo Efficacy of Multiple Doses ofIMGN779 (QWx 3) in MV4-11 Disseminated Model

[0402] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a disseminated tumor model was used as described in the protocol below.

[0403] Female NOD SCID mice were pre-treated with 150 mg/kg cyclophosphamide to partially ablate bone marrow in order to improve the engraftment of MV4-11 cells. The cyclophosphamide (Baxter, Lot # 4E011, Exp. 05/2017) was reconstituted with 0.9% NaCl and was administered intraperitoneally to the mice on day -3 and day -2 prior to MV4-11 cell inoculation on day 0. Following cyclophosphamide treatment as described above, the mice were each injected intravenously in the lateral tail vein with 3x106 MV4- 11 cells, a human AML cell line, in 100 μΐ of serum-free medium. On day 20 post-MV4- 11 inoculation, mice were randomized into the study groups. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 AML cells, preventing non-specific up-take of conjugate. Similarly, on days 27 and 34 post-MV4-l 1 inoculation, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 AML cells. On days 21, 28 and 35 post-MV4-l 1 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 μg/kg (by DGN462; 0.534 mg/kg by antibody) IMGN779, 3 μg/kg (by DGN462; 0.160 mg/kg by antibody) IMGN779, 1 μg/kg (by DGN462; 0.0534 mg/kg by antibody) IMGN779, 0.3 μg/kg (by DGN462; 0.0160 mg/kg by antibody) IMGN779, 0.1 μg/kg (by DGN462; 0.00534 mg/kg by antibody) IMGN779, 0.3 μg/kg (by DGN462; 0.00160 mg/kg by antibody) IMGN779 or 10 μg/kg (by DGN462; 0.556 mg/kg by antibody) ch TI-s-SPDB-DGN462.

[0404] Results are summarized in Table 13 (below) and in FIG. 13. The 10 μg/kg and 3 μg/kg doses of IMGN779, each administered qw x 3, were both highly active, generating a 108% and a 73% ILS, respectively, with corresponding tumor growth delays of 52 days and 35 days, respectively. The regimen of 1 μg/kg of IMGN779, qw x 3, was the reliable minimum effective dose in this study, yielding a 33% ILS and a 16-day tumor growth delay. The lower doses of IMGN779: 0.3, 0.1 and 0.03 μg/kg, qw x 3, resulted in ILS of 15%, 29% and 33%, respectively, and tumor growth delays of 15 days, 29 days and 33 days, respectively. The 10 ug/kg dose of the non-targeted Ab-DGN462 conjugate was also highly active, but yielded only a 61.5% ILS and a 29.5 tumor growth delay, which, when compared to the 108% ILS and 52 day tumor growth delay, demonstrates the CD33-targeted advantage of IMGN779.

TABLE 13:

Example 12. In Vivo Efficacy of Single Doses ofIMGN779 or Z6481A-S-SPDB- DGN484 in MV4-11 Disseminated Model

[0405] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a disseminated tumor model was used as described in the protocol below.

[0406] Female NOD SCID mice were pre-treated with 150 mg/kg cyclophosphamide to partially ablate bone marrow in order to improve the engraftment of MV4-11 cells. The cyclophosphamide (Baxter, Lot # 4E011, Exp. 05/2017) was reconstituted with 0.9% NaCl and was administered intraperitoneally to the mice on day -3 and day -2 prior to MV4-11 cell inoculation on day 0. Following cyclophosphamide treatment as described above, the mice were each injected intravenously in the lateral tail vein with 3x106 MV4- 11 cells, a human AML cell line, in 100 μΐ of serum-free medium. On day 7 post-MV4- 11 inoculation, mice were randomized into the study groups. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 AML cells, preventing non-specific up-take of conjugate. Similarly, on days 11 and 16 post-MV4-l 1 inoculation, the mice were injected intraperitoneally with 100 mg/kg of non-targeted chKTI antibody to block Fc receptors on the MV4-11 AML cells. On day 7 post-MV4-l 1 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 2.5 g/kg (by DGN462; 0.130 mg/kg by antibody) IMGN779, 1 μg/kg (by DGN462; 0.0521 mg/kg by antibody) IMGN779 or 0.5 μg/kg (by DGN462; 0.0260 mg/kg by antibody) IMGN779, 2.5 μg/kg Z4681A-S-SPDB-DGN484 (by DGN484; 0.135 mg/kg by antibody), 1 μg/kg (by DGN484; 0.0541 mg/kg by antibody) Z4681A-S-SPDB-DGN484, 0.5 μg/kg (by DGN484; 0.0271 mg/kg by antibody) Z4681A-S-SPDB-DGN484, 0.25 μg/kg (by

DGN484; 0.0135 mg/kg by antibody) or 0.1 μg/kg (by DGN484; 0.00541 mg/kg by antibody).

[0407] Results are summarized in Table 14 (below) and in FIG. 14. The 2.5 μg/kg, the

1.0 μg/kg and the 0.5 μg/kg doses (by DGN462) of IMGN779 were all highly active, generating Tumor Growth Delay (T-C) values of 32.5 days, 44 days and 56.5 days, respectively, and a % Increased Life Span (ILS) of 64, 87 and 112, respectively. The 2.5 μg/kg, the 1 μg/kg, the 0.5 μg/kg, the 0.25 μg/kg and the 0.1 μg/kg doses (by DGN484) of Z4681A-S-SPDB-DGN484 were all highly active, generating Tumor Growth Delay values of 38.5 days, 46.5 days, 26.5 days, 49 days and 34.5 days, respectively, and a %ILS of 76, 92, 52, 97 and 68, respectively. TABLE 14:

Example 13. In Vivo Efficacy of Multiple Doses ofIMGN779 (QWx 3) in Molm-13 Disseminated Model

[0408] To test the efficacy of IMGN779 for the ability to decrease tumor burden in vivo, a disseminated tumor model was used as described in the protocol below.

[0409] Female NOD SCID mice were pre-treated with 150 mg/kg cyclophosphamide to partially ablate bone marrow in order to improve the engraftment of Molm-13 cells. The cyclophosphamide (Baxter, Lot # 4E011, Exp. 05/2017) was reconstituted with 0.9% NaCl and was administered intraperitoneally to the mice on day -2 prior to Molm-13 cell inoculation on day 0. Following cyclophosphamide treatment as described above, the mice were each injected intravenously in the lateral tail vein with 2x105 Molm-13 cells, a human AML cell line, in 100 μΐ of serum-free medium. On day 6 post-Molm-13 inoculation, mice were randomized into the study groups by body weight. At 24 h prior to conjugate administration, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the Molm-13 AML cells, preventing non-specific up-take of conjugate. Similarly, on days 13 and 20 post-Molm- 13 inoculation, the mice were injected intraperitoneally with 400 mg/kg of non-targeted chKTI antibody to block Fc receptors on the Molm-13 AML cells. On days 7, 14 and 21 post-Molm-13 inoculation, the mice received a single intravenous injection, in the lateral tail vein, of vehicle, 10 (by DGN462; 0.534 mg/kg by antibody) IMGN779, 3 μg/kg (by DGN462; 0.160 mg/kg by antibody) IMGN779, 1 μg/kg (by DGN462; 0.0534 mg/kg by antibody) IMGN779, 0.3 μg/kg (by DGN462; 0.0160 mg/kg by antibody) IMGN779, 0.1 μg/kg (by DGN462; 0.00534 mg/kg by antibody) IMGN779, 10 μg/kg (by DGN462; 0.556 mg/kg by antibody) chKTI-s-SPDB-DGN462 or 3 μg/kg (by DGN462; 0.167 mg/kg by antibody) chKTI-s-SPDB-DGN462.

[0410] Results are summarized in Table 15 (below) and in FIG. 15. The 10, 3, 1 and 0.3 μg/kg doses of EVIGN779, each administered qw x 3, were all highly active, resulting in a >158%, a >158%, a 126% and a 58% ILS, respectively, and tumor growth delays of > 30 days, > 30 days, 24 days and 11 days, respectively. The 0.1 μg/kg dose of IMGN779, administered qw x 3, was the minimum effective dose and was minimally active, resulting in a 26% ILS and a 5-day tumor growth delay. In contrast, the 10 μg/kg and the 3 μg/kg doses of the non-targeted control conjugate, chKTI-s-SPDB-DGN462, each administered qw x 3, were both inactive, resulting in a 16% and a 24% ILS, respectively, and a 3-day and a 4.5-day tumor growth delay, respectively, demonstrating the CD33- targeting specificity of IMGN779.

TABLE 15:

Example 14. A Phase 1 Study of IMGN779 Administered Intravenously in Adult Patients with Relapsed/Refractory CD33-positive Acute Myeloid Leukemia

[0411] An open label, multi center Phase 1 study to determine the maximum tolerated dose (MTD), dosing schedule, and recommended Phase 2 dose (RP2D) of IMGN779 when administered as monotherapy to adult acute myeloid leukemia (AML) patients with CD33-positive disease was initiated. The study is a 2-stage, first-in-human (FIH) study consisting of 1) a dose escalation stage to identify the MTD and dosing schedule; and 2) a dose expansion stage. Two dosing schedules will be evaluated: Q2W (Schedule A: IMGN779 administered IV on days 1 and 15 of a 28-day cycle) and QW (Schedule B: IMGN779 administered IV on days 1 , 8, 15 and 22 of a 28-day cycle).

[0412] The patient population enrolled in the dose escalation stage will include adult (at least 18 years of age) patients with CD33 -positive, relapsed or refractory AML (with at least 20% of blasts being CD33 -positive, as determined by flow cytometry performed at a local CLIA-certified laboratory). Relapsed/ refractory AML is defined as 1) primary induction failure after 2 or more cycles of chemotherapy; 2) first early relapse after a remission duration of fewer than 6 months; and 3) second or subsequent relapse. The primary aim of the dose escalation stage is to identify the MTD and recommended schedule for IMGN779.

[0413] In some instances, patient blood samples or buccal cell swabs are collected and assessed for single nucleotide polymorphisms (S P) in the CD33 gene (Table 16) using technologies known in the art, including, but not limited to polymerase chain reaction (PCR), Sanger sequencing; next generation sequencing (NGS); reverse transcriptase quantitative polymerase chain reaction (RT-qPCR or qPCR) or similar technologies known to the art to identify DNA sequence. Patients may also be selected through determination of the levels and or ratios of the various splice variants (see, Lazlo et al. Oncotarget, Vol. 7, No. 28) of CD33 mRNA (full length (FL), delta-exon 2(ΔΕ2) delta- exon7a (E7a) and delta-exon2 and delta exon7a (ΔΕ2- E7a) using technologies known in the art, including, but not limited to, quantitative polymerase chain reaction (RT-qPCR or qPCR) or RNA-sequencing by Next Generation sequencing (NGS).

[0414] The dose escalation stage will follow a 3+3 cohort design with each cohort

enrolling 3 to 6 patients. Study enrollment will open with IMGN779 being administered on Schedule A (Q2W) at the starting dose of 0.02 mg/kg (by huMy9-6), which is l/6th of the HNSTD determined by a repeat-dose GLP toxicity study in cynomolgus monkeys. Dose escalation on this schedule will follow a modified Fibonacci dosing schema.

[0415] The Cohort Review Committee (CRC) will convene after each cohort to review the collected safety, PK Pd, and anti-tumor activity data; based on these data, the CRC may decide to continue dose escalation in Schedule A, add additional patients at the same dose level, or stop enrollment in that dose level or schedule. The CRC may also recommend opening the weekly dosing Schedule B. The starting dose for Schedule B will be determined by the CRC based on safety, Pd, and PK data, with a maximum of 1 dose level below the last dose level that was cleared by the CRC from Schedule A. Dose escalation will proceed in Schedule A as shown in Table 17 until an MTD is defined.

[0416] The MTD for a given schedule will be determined from the assessment of Dose

Limiting Toxicities (DLTs) during the first dosing cycle. Clinically significant toxicities or treatment-emergent adverse events (TEAEs) that meet the definition of dose limiting but occurring after Cycle 1 may be considered when determining the MTD for each schedule. The use of hydroxyurea or leukapheresis during the study will be taken into consideration as it may be a confounding factor.

[0417] To better characterize safety, up to 12 patients may receive EVIGN779 at the

putative RP2D in each dosing schedule, based on the recommendation of the CRC and drug sponsor. The CRC will convene to review the available safety, PK/Pd, and antitumor activity data and select the dose and dosing schedule to be used during the dose expansion portion of the study. Given that IMGN779 is a targeted therapy, available information about the target along with safety, PK, and Pd data will be considered when selecting the recommended dose for the expansion phase.

[0418] Patients enrolled in the dose expansion stage will be assigned to a cohort as

follows: patients with CD33-positive AML at first relapse (disease that has relapsed more than 6 months after initial remission). AML at first relapse is defined as disease that has relapsed more than 6 months after initial remission. During the dose expansion stage of the study, a Bayesian continual reassessment method (CRM) approach will be used to guide the monitoring of DLTs. Once the putative RP2D and schedule are selected, the dose expansion stage of the study will open. The dose expansion portion of the study is designed to further evaluate safety and tolerability, assess anti-tumor activity at the putative RP2D, and characterize IMGN779 PK. Data obtained in the dose expansion phase will help refine the RP2D.

TABLE 16:

TABLE 17:

[0419] Secondary objectives include assessment of IMGN779 safety and tolerability, characterization of the PK profile of EVIGN779, evaluation of potential immunogenicity, and assessment of anti-tumor activity. Secondary endpoints include identification of treatment emergent adverse events, PK parameters such as Cmax, AUC, and terminal half-life (t½), human anti-drug antibody levels, objective response rate (ORR), overall survival, relapse free survival (RFS), event free survival (EFS), and cumulative incidence of relapse (duration of remission) at 6 and 12 months.

[0420] The period of observation extends from the time the patient receives the first dose of IMGN779 until the final follow-up study visit. Patients will continue to receive IMGN779 until unacceptable toxicity or withdrawal of consent, whichever comes first, or until the drug sponsor terminates the study. Patients who discontinue study drug for reasons other than progressive disease will be followed until progressive disease, start of new anti-cancer therapy, or death, whichever occurs first.

[0421] PK parameters such as Cmax, Tmax, AUC, ΐ1/2(β), volume of distribution at steady state (Vss), clearance (CL), will be derived as is feasible from plasma

concentrations of IMGN779, total Z4681A antibody and DGN462, and potential metabolites using the actual sampling times when possible. In cases where actual sampling times are missing, nominal times will be used. Concentration data and PK parameters will be summarized descriptively by dose. Non-compartmental methods will be used to compute the PK parameters using SAS and/or Phoenix WinNonlin software. Plasma concentration time profiles will be presented graphically by dose level.

[0422] Blood samples will be taken to assess the PK properties of IMGN779 when

administered IV once every 2 weeks (Schedule A) or weekly (Schedule B). Blood samples for PK measurements will be taken, for all patients, at the following time-points, and at unscheduled visits, end of treatment visit, and 30-day follow up visit:

Schedule A (Q2W)

• Cycles 1 and 3

o Day 1 - pre-dose and immediately following the completion of infusion of IMGN779 (+5 minutes); and at 2 hours (±10 minutes), 4 hours (±15 minutes), and 6 hours (±1 hour) after the completion of the EVIGN779 infusion

o Day 2 - 24 hours after completion of EVIGN779 infusion (±2 hours) o Day 3 - 48 hours after completion of IMGN779 infusion (±4 hours) o Day 8 - a single blood sample

o Day 15 - pre-dose and immediately following the completion of infusion of IMGN779 (+5 minutes)

• Cycle 2 and Cycles 4 to 6

o Day 1 - pre-dose and immediately following the completion of infusion of

IMGN779 (+5 minutes)

Schedule B (QW)

• Cycle 1 o Day 1 - pre-dose and immediately following the completion of infusion of IMGN779 (+5 minutes); and at 2 hours (±10 minutes), 4 hours (±15 minutes), and 6 hours (±1 hour) after the completion of the IMGN779 infusion

o Day 2 - 24 hours after completion of IMGN779 infusion (±2 hours) o Day 3 - 48 hours after completion of IMGN779 infusion (±4 hours) o Day 8 - pre-dose and immediately following the completion of infusion of

IMGN779 (+5 minutes)

o Day 22 - pre-dose and immediately following the completion of infusion of IMGN779 (+5 minutes)

• Cycles 2 to 6

o Day 1 - pre-dose, and immediately following the completion of infusion of

IMGN779 (+5 minutes)

o Day 8 - pre-dose, and immediately following the completion of infusion of

IMGN779 (+5 minutes)

Example 15. IMGN779 PK Exposure Metrics

[0423] Nineteen patients with a median age of 62 received six dose levels ranging from

0.02 to 0.39 mg/kg IMGN779. Pharmacokinetic (PK) parameters were determined by noncompartmental analysis (NCA) of plasma samples taken at time points as outlined above in Example 14.

[0424] Adverse events (AE) were as expected for this refractory AML population

including cytopenias and constitional symptoms. No relationship between frequency or severity and IMGN779 dose level was observed. The most common AEs were nausea (41%), febrile neutropenia (29%), and rash (29%). Pneumonia, respiratory failure, and constitpation were additional AEs reported in 4 or more patients (> 24%). The most common serious adverse events (SAEs) were grade 3 febrile nuetropenia (29%) and pneumonia (24%). No dose limiting toxicities have been reported. Importantly, no safety signals regarding liver or hematopoietic toxicity have been observed in laboratory assessments.

[0425] As the clinical trial continued, a total of twenty-three patients (including the 19 patients discussed above) with a median age of 68 received seven dose levels ranging from 0.02 to 0.54 mg/kg IMGN779.

TABLE 18:

[0426] The demographics of these twenty-three patients are shown in FIG. 16. The

treatment-emergent adverse events are shown in FIGs. 17 and 18. The most frequent adverse events were:

• Cytopenias: febrile neutropenia (39%), anemia (17%), and thrombocytopenia (13%);

• Gastrointenstinal: constipation (22%), diarrhea (22%), and abdominal pain (17%); and

• Respiratory: dyspnea (22%), pneumonia (22%), and respiratory failure (17%).

[0427] The most frequent Grade 3 or more adverse events were:

• Cytopenias: febrile neutropenia (39%), anemia (17%), and thrombocytopenia (9%); and

• Respiratory: pneumonia (22%) and respiratory failure (17%).

[0428] Other adverse events of interest included infusion reactions (4%), bilirubin (TBili) elevation (8%), and alanine transaminase (ALT) elevation (4%). The most frequent serious adverse events (SAEs) included febrile neutropenia (39%), pneumonia (17%), and respiratory failure (17%). Grade 3 infusion reactions were related serious adverse events.

[0429] No dose limiting toxicities were observed at doses up to 0.54 mg/kg (cohort 7). In addition, no evidence of cumulative toxicity was observed with up to 17 doses given per patient.

[0430] Two of three patients treated with IMGN779 at 0.54 mg/kg (cohort 7) cleared their peripheral blasts, and the third demonstrated a 66% reduction of peripheral blasts. One patient treated at 0.39 mg/kg (cohort 6) had a <50% reduction in bone marrow blasts from 54% to 25% after one cycle.

[0431] Additional anti-leukemic activity observed in cohorts 6 and 7 is shown in FIGs.

19 and 20. FIG. 19 shows the effect of IMGN779 on peripheral blood blasts. All patients in cohorts 6 and 7 showed a decrease from baseline in peripheral blasts within the first 3-8 days of the first dose. The baseline is the peripheral blood blasts count at day 0 (i.e., prior to administration) for each patient. For the three patients whose peripheral blood blasts progressed after day 3-8, all showed decreased blasts after the second dose (day 15).

[0432] FIG. 20 shows the effect of IMGN779 on bone marrow blasts. Three patients from cohorts 5 and 6 showed at least a 45% decrease from baseline in bone marrow blasts (without hydroxyurea). One patient had a reduction from 90% to 4% (96% reduction from baseline). Another had a reduction from 54% to 22% (59% reduction from baseline), and a third had a reduction from 23% to 12% (48% reduction from baseline). The baseline is the bone marrow blasts count at day 0 (i.e., prior to administration) for each patient.

[0433] The pharmacokinetic (PK) parameters are reported for Cycle 1 (first cycle of dosing for each patient only) of the IMGN779 Phase 1 trial (see FIGs. 21A-21C and Table 19). The exposure of IMGN779 is shown to be minimal at low doses with insufficient data to calculate the terminal elimination half-life. BVIGN779 exposures tended to increase at higher doses, with a mean (± SD) half-life of 18.3 ± 10.2 hr at the 0.26 mg/kg dose level. Overall, the exposure (AUC) and the Cmax are shown to generally increase at the higher doses with a linear relationship between the Cmax and dose at doses greater than 0.16 mg/kg and a non-linear relationship between the exposure and dose (see FIGs. 21B and 21C). IMGN779 is well tolerated at cohort 5 and 6 (doses of 0.26 mg/kg and 0.39 mg/kg, respectively). All three patients in cohort 6 had sustained exposure observed through 168 hours post infusionand had sustained decrease of CD33 levels through day 8.

[0434] The pharmacokinetic and pharmacodynamic parameters for the patients in cohorts

4-7 are shown in FIG. 22. These results demonstrate that Cmax and AUC increased with dose and that cohorts 6 and 7 demonstrate prolonged exposure through 7 days from infusion. The duration of CD33 saturaction increased with dose, and cohorts 6 and 7 demonstrated prolonged CD33 saturation through 7 days from infusion. [0435] IMGN779 pharmacokinetics will continue to be evaluated and any association between clinical activity and EVIGN779 pharmacokinetics will be characterized.

[0436] A pharmacodynamics (PD) assay to measure the saturation of residual free-CD33 was used to demonstrate that in cohort 5 there was consistent saturation of free CD33 in all patients past 48 hours, consistent with the PK results.

[0437] Without wishing to be bound by theory, at similar doses of -0.26 mg/kg,

gemtuzumab ozogamicin Mylotarg®) exposure as indicated by the calculated Cmax is higher than EVIGN779, suggesting that the difference could be in the Kds of the antibodies (i.e., the Z4681A antibody comprised in IMGN779 has a higher affinity to CD33 than the hP67.6 antibody comprised in gemtuzumab ozogamicin).

TABLE 19:

Example 16. IMGN779 Exposure and Efficacy

[0438] As the clinical trial continued, a total of thirty-six patients (including the 23

patients discussed above) with a median age of 67 received dose levels ranging from 0.02 to 0.91 mg/kg IMGN779.

TABLE 20:

[0439] Continued increases in maximal concentration and exposure (AUCias_t) were

observed up to doses of 0.91 mg/kg Q2W, and an increased duration of CD33 saturation also continued to be observed with increased dosing (FIG. 23).

[0440] In addition to dosing every other week (Q2W), IMGN779 was also administered weekly (QW) at a dose of 0.39 mg/kg. Weekly dosing increased the duration of CD33 saturation as compared to Q2w dosing, but no substantial difference in accumlation of IMGN779 was observed with weekly dosing as compared dosing Q2w (FIG. 24).

[0441] Furthermore, sixteen out of seventeen evaluable patients showed a decrease in peripheral blood blasts within ten days of the first dose. The median maximal decrease was 71%, with a range of 16% to 100%. In addition, seven of the seventeen evaluable patients showed a 48%-96% reduction in bone marrow blasts (FIG. 25). Of these seven patients, six had prior intense treatment, three had primary-refractory disease, three had mutations in RAS, two had mutations in TP53, two had mutations in IDH, and two had mutations in FLT3. In addition, one patient treated at a lower dose (0.16 mg/kg Q2W) showed a 93% reduction (48% to 3%) reduction in bone marros blasts with extended treatment (Cycle 5) and continued on through Cycle 14. Thus, IMGN779 reduces peripheral blood blasts in AML patients.

Example 17. Continued Evaluation of IMGN779 Safety, Exposure Metrics, and Efficacy

[0442] As the clinical trial further progressed, three patients were administered 1.2 mg/kg

IMGN779 every other week (Q2W), and three other patients were administered 1.5 mg/kg IMGN779 on the same Q2W schedule. Six patients were also administered 0.54 mg/kg IMGN779 every week (QW).

[0443] No DLTs were observed for any patients enrolled in this study (including the patients described in Examples 15 and 16), and no patterns of IMGN779 toxicity emerged. As shown in FIG. 26, less than 40% of patients exhibited febrile neutropenia. Moreover, only one patient experienced a Grade 3+ TEAE (hyperbilirubinaemia), and only two patients experienced a suspected unexpected serious adverse reaction (SUSAR) (one due to Grade 3 infusion reaction and one due to colonic perforation). Data regarding hepatoxicity was anecdotal, with two patients exhibiting Grade 3 increased total bilirubin (TBili), one patient exhibiting Grade 3 alanine transaminase (ALT) elevation, and one patient exhibiting Grade 3 aspartate transaminase (AST). Each of these Grade 3 events was observed in a different patient, and no Hy's Law cases were observed based on drug- induced liver injury assessment.

[0444] No patterns of neutropenia or thrombocytopenia were observed across the various patient cohorts, although several of patients administered 0.54 mg/kg IMGN779 on a QW schedule exhibited cytopenias. In particular, one patient had significant decreases in neutrophils, hemoglobin, and platelets during cycle 1 in the setting of an SAE for diverticulitis. Although the SAE resolved, and the patient had a decrease in bone marrow (BM) blasts (-78%, 27% to 6%), the patient's counts did not recover during 4 weeks of observation. It was unclear whether the cytopenia observed in this patient was an effect of treatment with IMGN779. Additionally, three other patients had moderate to significant decreases in neutrophils and/or platelet counts, but all of these patients had progressing AML as a common confounder. One patient also had moderate increases in platelets and stable neutrophil counts in the setting of a modest decrease in BM blasts (-30%, 26% to 18%). Collectively, none of these observations met the definition of a DLT.

[0445] Pharmacokinetic and pharmacodynamic data related to IMGN779 concentration and CD33 saturation were collected throughout the study. As shown in FIG. 27A, the maximal concentration and exposure of IMGN779 continued to increase with dose amount in Cycle 1 of treatment. As shown in FIG. 27B, upon repeat dosing, the QW dose schedule was associated with slightly higher end of infusion concentrations compared to the Q2W dose schedule, but there was no substantial accumulation of IMGN779. As shown in FIGs. 28A and 28B, increased CD33 saturation (i.e., lower CD33 receptor availability on blasts) was observed with increased dosing. Saturation was not maintained between doses in the Q2W schedule (FIG. 28A), but saturation was maintained between doses in the QW schedule (FIG. 28B). As shown in FIGs. 29A and 29B, complete saturation occurred with QW dosing at 0.54 mg/kg (FIG. 29B), but incomplete saturation was observed for Q2W dosing at the same dose amount (FIG. 29A). Collectively, these data indicate that exposure metrics (e.g., Cmax and AUC) increase with dose amount and that CD33 saturation is prolonged in the QW dosing schedule.

[0446] To assess whether CD33 SNPs are associated with response to IMGN779

treatment, the level of CD33 expression on patient cells was determined and further characterized by SNP allele combinations. As shown in FIGs. 30A and 30B, uniform CD33 expression across blasts appeared to be a better indicator of a response than the amount of CD33 on each blast. Moreover, the majority of non-formal responders had high CD33% positivity. Previous studies with gemtuzumab ozogamicin have suggested a particular therapeutic benefit in pediatric AML patients with the rsl2459419 SNP (associated with the CC allele); however the same trend was not observed in previous MRC adult-AML clinical trials. In the present study with adult-AML patients, a clinical benefit of IMGN779 was observed for all three possible SNP allele combinations (CC, CT, and TT), as shown in FIG. 31. Accordingly, these data indicate that the CD33 T allele does not preclude a response to IMGN779 and that a particular CD33 SNP does not provide a definitive prediction of response.

[0447] Finally, the best response (measured as decreased bone marrow blast %) was observed across all patient cohorts in the study. While only one patient had a complete remission with incomplete hematologic recovery (CRi), a significant fraction of patients (-44%, 11 out of 25) demonstrated signs of efficacy. As shown in FIG. 32, efficacy signals were seen in both the Q2W and QW dosing schedules. Collectively, these data suggest a broad activity of IMGN779 in a relapsed/refractory AML population.

Example 18. Pharmacokinetic Modelling of IMGN779

[0448] Pharmacokinetic (PK) modelling was used to assess the effect of IMGN779 dose on elimination half-life. A clear dose effect on elimination half-life was observed in patients receiving IMGN779 and it was seen that elimination half-life was slower with higher doses. In particular, IMGN779 blood concentrations rapidly dropped by 10-20 nM in all low dose subjects. The same rapid drop was also observed in some individuals receiving higher doses, followed by a slower elimination phase. The rapid elimination of EVIGN779 observed in low dose subjects (i.e., resulting in a final IMGN779 blood concentration of <10 nM) but not in the highest dose subjects indicated that there was no consistent non-linear concentration range. A simple one-compartment PK model was then applied to probe the data and estimate individual concentration - time slopes. As shown in FIG. 33, the dose dependency was confirmed in slope estimates. However, as shown in FIG. 34, a typical linear PK model that does not account for changes in target or dose did not fit well with the data.

[0449] Data from patients receiving 0.54 mg/kg EVIGN779 were then used to assess correlations of elimination rate constant with the number of binding sites per cell and the total target concentration. As shown in FIGs. 35A and 35B, elimination rate constants did not correlate with the number of binding sites (FIG. 35A) or the total target concentration (FIG. 35B). A target mediated drug disposition (TMDD) model was then used to assess linear + target mediated elimination. Because only total target (R + RL) was measured, instant binding was assumed. To calculate measured target, the number of binding sites per cell (ABC) was determined for monocytes, lymphocytes, blasts, and granulocytes. Measured target (i.e., ABC* cell count (n/L)) was calculated in circulating cells for each cell type and added up to a total. As shown in FIG. 36, TMDD based on measured target levels did not fit any better than the linear PK model. Accordingly, similar to the linear model,adding a target mediated elimination model using measured target could not explain the observed PK profiles.

[0450] A simple binding model was then applied to the data, in which estimated target levels are not dependent on measured target levels and there is no target mediated elimination (only saturable binding). Target Concentration Pool was estimated at 16.4 nM and compared to a measured target concentration value of 0.02 nM. As shown in FIGs. 37 A and 37B, no obvious trends were observed between estimated target and individual measured circulating target concentrations, indicating that the pool of target / non-specific binding needed to explain PK is outside of the blood. Although not a perfect fit, this binding model was associated with an increased fit to the data compared to a linear PK model or a TMDD model using measured target (FIG. 38).

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject comprising administering to the subject (i) about 0.54 to about 0.8 mg/kg (ii) about 0.30 to about 0.54 mg/kg (iii) about 0.7 to about 1 mg/kg (iv) about 0.39 to about 0.7 mg/kg (v) about 0.7 to about 1.25 mg/kg or (vi) about 1 to about 2 mg/kg of an anti-CD33 immunoconjugate of the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line— between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is hydrogen, when it is a single bond, X is hydrogen and Y is -S0₃H;

r is an integer from 1 to 10; and

A is an anti-CD33 antibody or antigen-binding fragment thereof comprising a variable heavy chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1-3, respectively and a variable light chain comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, respectively.

2. The method claim 1, wherein about 0.54 to about 0.70 mg/kg of the immunoconjugate is administered.

3. The method of claim 1, wherein about 0.54 or about 0.70 mg/kg of the immunoconjugate is administered.

4. The method of any one of claims 1-3, wherein the immunoconjugate is administered about once in two weeks.

5. The method of claim 1, wherein about 0.39 to about 0.54 mg/kg of the immunoconjugate is administered.

6. The method of claim 1, wherein about 0.39 mg/kg or about 0.54 mg/kg of the

immunoconjugate is administered.

7. The method of any one of claims 1, 5, and 6, wherein the immunoconjugate is

administered about once a week.

8. The method claim 1, wherein about 0.7 to about 0.9 mg/kg of the immunoconjugate is administered.

9. The method of claim 1, wherein about 0.7 mg/kg, about 0.75 mg/kg, about 0.8 mg/kg, about 0.85 mg/kg, about 0.9 mg/kg, or about 1 mg/kg of the immunoconjugate is administered.

10. The method of any one of claims 1, 8, and 9, wherein the immunoconjugate is

administered about once in two weeks.

1 1. The method of claim 1, wherein about 0.39 mg/kg, about 0.45 mg/kg, about 0.5 mg/kg, about 0.54 mg/kg, about 0.55 mg/kg, about 0.6 mg/kg, about 0.65 mg/kg, or about 0.7 mg/kg of the immunoconjugate is administered

12. The method of claim 1 or 11, wherein the immunoconjugate is administered about once a week.

13. The method of claim 1, wherein about 0.7 to about 1 mg/kg or about 0.7 to about 0.9 mg/kg of the immunoconjugate is administered.

14. The method of claim 1, wherein about 0.7 mg/kg, about 0.75 mg/kg, about 0.8 mg/kg, about 0.85 mg/kg, about 0.9 mg/kg, about 0.95 mg/kg, or about 1 mg/kg of the immunoconjugate is administered.

15. The method of claim 1, 13, or 14, wherein the immunoconjugate is administered about once a week.

16. The method claim 1, wherein about 1 to about 1.25 mg/kg of the immunoconjugate is administered.

17. The method of claim 1, wherein about 1.1 mg/kg, about 1.15 mg/kg, about 1.2 mg/kg, or about 1.25 mg/kg, of the immunoconjugate is administered.

18. The method of any one of claims 1, 16, and 17, wherein the immunoconjugate is

administered about once in two weeks.

19. The method of any one of claims 1, 16, and 17, wherein the immunoconjugate is

administered about once a week.

20. The method claim 1, wherein about 1.2 to about 2 mg/kg or about 1.5 to about 1.6 mg/kg of the immunoconjugate is administered.

21. The method of claim 1, wherein about 1.5 mg/kg or about 1.56 mg/kg of the

immunoconjugate is administered.

22. The method of any one of claims 1, 20, and 21, wherein the immunoconjugate is

administered about once in two weeks.

23. The method of any one of claims 1, 20, and 21, wherein the immunoconjugate is

administered about once a week.

24. The method of any one of claims 1-23, wherein the immunoconjugate is administered on a 28-day cycle.

25. The method of any one of claims 1-24, wherein the immunoconjugate is administered by intravenous infusion.

26. The method of any one of claims 1-25, wherein the variable heavy chain comprises the sequence set forth in SEQ ID NO:9 and the variable light chain comprises the sequence set forth in SEQ ID NO: 10.

27. The method of claim 26, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 11 and a light chain comprising the sequence set forth in SEQ ID NO: 12.

28. The method of any one of claims 1-27, wherein the immunoconjugate is an

immunoconjugate of Formula (II):

29. The method of any one of claims 1-27, wherein the immunoconjugate is an

immunoconjugate of Formula (III):

30. The method of any one of claims 1-29, wherein the pharmaceutically acceptable salt is a sodium or potassium salt.

31. The method of any one of claims 1-30, wherein r is an integer from 2 to 9, from 3 to 8, from 4 to 7, or from 5 to 6.

32. The method of any one of claims 1-30, wherein the immunoconjugate is contained in a composition comprising at least two of the immunoconjugates and wherein the average number of cytotoxins per antibody or antigen-binding fragment thereof is 2 to 8, 3 to 7, 3 to 5 or 2.5 to 3.5.

33. The method of any one of claims 1-32, wherein the immuoconjugate is IMGN779.

34. The method of any one of claims 1-33, wherein the cancer is selected from the group consisting of leukemia, lymphoma and myeloma.

35. The method of claim 34, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell lineage acute lymphoblastic leukemia (B ALL), T-cell acute lymphoblastic leukemia (T ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS), basic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin lymphomas (NHL), mantle cell lymphoma, and Hodgkin's leukemia (HL).

36. The method of claim 35, wherein the cancer is acute myeloid leukemia (AML).

37. The method of claim 36, wherein the AML is refractory or relapsed acute myeloid

leukemia.

38. The method of claim 36, wherein the AML is newly diagnosed.

39. The method of any one of claims 36-38, wherein the AML is characterized by

overexpression of P-glycoprotein, overexpression of EVI1, a p53 alteration, D MT3A mutation, FLT3 internal tandem duplication, a complex karyotype, decreased expression in BRCA1, BRCA2, or PALB2, or mutations in BRCA1, BRCA2, or PALB2.

40. The method of claim 35, wherein the MDS is high risk MDS.

41. The method of any one of claims 1-40, wherein a homozygous rsl2459419C genotype has been detected in a sample obtained from the subject.

42. The method of claim 41, further comprising detecting the homozygous rsl2459419C genotype in a sample obtained from the subject.

43. The method of any one of claims 1-40, wherein a heterozygous rsl2459419C genotype has been detected in a sample obtained from the subject.

44. The method of claim 43, further comprising detecting the heterozygous rsl2459419C genotype in a sample obtained from the subject.

45. The method of any one of claims 1-40, wherein a homozygous rsl 2459419T genotype has been detected in a sample obtained from the subject.

46. The method of claim 45, further comprising detecting the homozygous rsl2459419T genotype in a sample obtained from the subject.

47. The method of any one of claims 1-40, wherein a heterozygous rsl2459419T genotype has been detected in a sample obtained from the subject.

48. The method of claim 47, further comprising detecting the heterozygous rsl2459419T genotype in a sample obtained from the subject.

49. The method of any one of claims 41-48, wherein the sample obtained from the patient is a blood sample or a buccal swab.

50. The method of any one of claims 149, wherein the cancer is chemotherapy sensitive.

51. The method of claims 1-49, wherein the cancer is chemotherapy resistant.

52. The method of any one of claims 1-51, wherein at least 20% of blasts from the cancer are CD33-positive as measured by flow cytometry.

53. The method of any one of claims 1-52, wherein the administration results in saturation of free-CD33.

54. The method of any one of claims 1-53, wherein the administration results in a decrease in peripheral blood blasts.

55. The method of any one of claims 1-54, wherein the administration results in a decrease in bone marrow blasts.

56. The method of any one of claims 1-55, wherein the subject is a human.

57. The method of any one of claims 1-56, wherein the cancer is chacterized by a RAS mutation.

58. The method of any one of claims 1-57, wherein the cancer is chacterized by a TP53 mutation.

59. The method of any one of claims 1-58, wherein the cancer is chacterized by an IDH mutation.

60. The method of any one of claims 1-59, wherein the cancer is chacterized by an FLT3 mutation.