Methods of treating cancer with nonfucosylated anti-CD70 antibodies

Abstract

The present disclosure provides methods of treating cancer, for example, myeloid malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), using non-fucosylated anti-CD70 antibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 954,904, filed December 30, 2019, and U.S. Provisional Patent Application No. 63 / 011,906, filed April 17, 2020, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 761682003140SEQLIST.TXT, Recorded: December 9, 2020, Size: 13KB).

[0003] The present invention relates to methods of treating cancer, such as myeloid malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), with non-fucosylated anti-CD70 antibodies. [Background technology]

[0004] CD70 is a member of the tumor necrosis factor (TNF) family of membrane-bound and secreted molecules expressed by a variety of normal and malignant cell types. The primary amino acid (AA) sequence of CD70 predicts a type II transmembrane protein with its carboxyl terminus exposed to the outside of the cell and its amino terminus found on the cytoplasmic side of the cell membrane (Bowman et al., 1994, J. Immunol. 152:1756-61; Goodwin et al., 1993, Cell 73:447-56). Human CD70 consists of a 20-AA cytoplasmic domain, an 18-AA transmembrane domain, and a 155-AA extracytoplasmic domain with two potential N-linked glycosylation sites (Bowman et al., supra; Goodwin et al., supra). Specific immunoprecipitation of cells expressing radiolabeled CD70 with anti-CD70 antibodies yields polypeptides of 29 and 50 kDa (Goodwin et al., supra; Hintzen et al., 1994, J. Immunol. 152:1762-73). A trimeric structure of CD70 has been predicted based on its homology to TNF-alpha and TNF-beta, particularly in structural chains C, D, H, and 1 (Petsch et al., 1995, Mol. Immunol. 32:761-72).

[0005] Original immunohistochemical studies demonstrated that CD70 is expressed on germinal center B cells and rare T cells in the tonsils, skin, and intestine (Hintzen et al., 1994, Int. Immunol. 6:477-80). Later, CD70 was reported to be expressed on the cell surface of recently antigen-activated T and B lymphocytes, and its expression declines after removal of antigen stimulation (Lens et al., 1996, Eur. J. Immunol. 26:2964-71; Lens et al., 1997, Immunology 90:38-45). Within the lymphoid system, activated natural killer cells (Orengo et al., 1997, Clin. Exp. Immunol. 107:608-13) and mature peripheral murine dendritic cells (Akiba et al., 2000, J. Exp. Med. 191:375-80) also express CD70. In nonlymphoid lineages, CD70 has been detected on medullary thymic epithelial cells (Hintzen et al., 1994, supra; Hishima et al., 2000, Am. J. Surg. Pathol. 24:742-46).

[0006] CD70 is not expressed on normal nonhematopoietic cells. Under physiological conditions, CD70 expression is largely restricted to recently antigen-activated T and B cells, and its expression is downregulated after antigen stimulation ceases. Evidence from animal models suggests that CD70 may contribute to immunological disorders, such as rheumatoid arthritis (Brugnoni et al., 1997, Immunol. Lett. 55:99-104), psoriatic arthritis (Brugnoni et al., 1997, Immunol. Lett. 55:99-104), and lupus (Oelke et al., 2004, Arthritis Rheum. 50:1850-60). In addition to its potential role in inflammatory responses, CD70 is also expressed on a variety of transformed cells, including lymphoma B cells, Hodgkin and Reed-Sternberg cells, malignant cells of neural origin, and some carcinomas. Studies have shown that stem cells from patients with acute myeloid leukemia (AML) and myelodysplastic diseases (MDS) express both CD70 and its receptor, CD27. Interaction between this ligand-receptor pair may promote the survival and proliferation of leukemic blast cells.

[0007] Monoclonal antibodies produced in mammalian host cells may have various post-translational modifications, including glycosylation. Monoclonal antibodies, such as IgG1, have an N-linked glycosylation site at asparagine 297 (Asn297) in each heavy chain (two per intact antibody). The glycan attached to Asn297 on antibodies is typically a complex, biantennary structure with little or no bisecting N-acetylglucosamine (bisecting GlcNAc), a small amount of terminal sialic acid, and variable amounts of galactose. The glycan also typically has a high level of core fucosylation. Reduction of core fucosylation in antibodies has been shown to alter Fc effector function, particularly Fc gamma receptor binding and ADCC activity. This observation has led to interest in engineering cell lines to produce antibodies with reduced core fucosylation.

[0008] Methods for engineering cell lines to reduce core fucosylation include gene knockout, gene knockin, and RNA interference (RNAi). In gene knockout, the gene encoding FUT8 (alpha 1,6-fucosyltransferase enzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residue from GDP-fucose to the 6-position of Asn-linked (N-linked) GlcNAc of N-glycans. FUT8 has been reported to be the only enzyme responsible for adding fucose to N-linked biantennary carbohydrates at Asn297. Gene knockin adds genes encoding enzymes such as GNTIII or Golgi alpha mannosidase II. Increasing the levels of such enzymes in cells diverts monoclonal antibodies from the fucosylation pathway (leading to reduced core fucosylation) and results in increased amounts of bisecting N-acetylglucosamine. RNAi also typically targets FUT8 gene expression, leading to reduced mRNA transcript levels or complete knockout of gene expression.

[0009] An alternative to engineering cell lines involves the use of small molecule inhibitors that act on enzymes in the glycosylation pathway. Inhibitors, such as castanospermine, act early in the glycosylation pathway to produce antibodies with immature glycans (e.g., high levels of mannose) and low fucosylation levels. Antibodies produced by such methods generally lack the complex N-linked glycan structures associated with mature antibodies. Small molecule fucose analogs can also be used to generate recombinant antibodies with complex N-linked glycans but reduced core fucosylation.

[0010] There is a need for anti-CD70 antibodies, for example, anti-CD70 antibodies with reduced core fucosylation, that can exert clinically useful cytotoxic, cytostatic, or immunomodulatory effects on CD70-expressing cells, particularly without exerting undesirable effects on non-CD70-expressing cells. Such compounds would be useful therapeutic agents for CD70-expressing cancers.

[0011] Myeloid malignancies include acute myeloid leukemia (AML), myeloproliferative disorders (MPDS), myelodysplastic syndromes (MDS), and myelodysplastic / myeloproliferative syndromes, all of which are clonal stem cell (HSC) or precursor malignant disorders (TIU et al., Leukemia, 21(8), 1648-57, 2007).

[0012] MDS encompasses multiple subtypes, including MDS with single-lineage dysplasia, MDS with ringed sideroblasts, MDS with multilineage dysplasia, MDS with excess blasts, MDS with isolated del(5q), and unclassified MDS (ARBER et al., Blood, Vol. 127, pp. 2391-405, 2016). MDS is characterized by ineffective hematopoiesis in one or more myeloid lineages. Early-stage MDS often demonstrates excessive apoptosis and hematopoietic cell dysplasia (CLAESSENS et al., Blood, Vol. 99, pp. 1594-601, 2002; CLASESSENS et al., Blood, Vol. 105, pp. 4035-42, 2005). In approximately one-third of MDS patients, this ineffective hematopoiesis precedes progression to secondary AML (sAML). Although some molecular events associated with specific MDS subtypes (ELBERT et al., Nature, 451(7176), 335-9, 2008) or disease transformation (BRAUN et al., Blood, 107(3), 1156-65, 2006) have been identified, the underlying molecular defects remain poorly understood. Apart from morphological features, no biological markers are currently available for early diagnosis and prognosis.

[0013] Acute myeloid leukemia (AML) is a malignant tumor of the myeloid lineage of white blood cells. This blood stasis is a disease of the blood and bone marrow that is usually fatal within weeks to months if left untreated. There are an estimated 30,000 cases of AML in the United States and 47,000 cases in the European Union (2010 prevalence data confirmed by Mattson-Jack, 2010). AML is the most common form of acute leukemia in adults (approximately 90%) and accounts for approximately 33% of new leukemia cases. The median age of patients diagnosed with AML is 67 years. In the United States, AML accounts for approximately 1.2% of cancer deaths.

[0014] AML causes nonspecific symptoms such as weight loss, fatigue, fever, and night sweats. AML is diagnosed by blood tests, bone marrow tests, and laboratory tests to determine the AML subtype and determine treatment decisions.

[0015] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0016]

[0010] Provided herein are methods of treating a CD70-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a nonfucosylated anti-CD70 antibody, which results in depletion of cancer cells in the subject but does not result in depletion of CD70+ regulatory T cells (CD70+ Tregs) in the subject, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, where the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Fc domain is an antibody effector domain that mediates one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain is an antibody effector domain that mediates ADCC. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the anti-CD70 antibody is borsetuzumab. In some embodiments, the antibody is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent or an immunomodulatory agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some embodiments, the method comprises administering a population of anti-CD70 antibodies, wherein each antibody in the population of anti-CD70 antibodies comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibodies are defined by the Kabat numbering scheme, and at least 50% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation.In some embodiments, at least 70% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 90% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the cancer is MDS. In some embodiments, the MDS is relapsed or refractory MDS. In some embodiments, the subject has experienced treatment failure after prior hypomethylating agent (HMA) therapy for MDS. In some embodiments, the cancer is AML. In some embodiments, the AML is relapsed or refractory AML. In some embodiments, the subject has received two prior treatment regimens to treat AML. In some embodiments, the subject has received three prior treatment regimens to treat AML. In some embodiments, at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cancer cells express CD70. In some embodiments, administering a nonfucosylated anti-CD70 antibody to a subject results in at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% depletion of cancer cells compared to the amount of cancer cells before administering the nonfucosylated anti-CD70 antibody to the subject.In some embodiments, administering a nonfucosylated anti-CD70 antibody to a subject results in depletion of CD70+ Tregs by about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.1% or less compared to the amount of CD70+ Tregs before administering the nonfucosylated anti-CD70 antibody to the subject. In some embodiments, one or more therapeutic effects are improved in the subject after administration of the nonfucosylated anti-CD70 antibody relative to baseline. In some embodiments, the one or more therapeutic effects are selected from the group consisting of objective response rate, duration of response, time to response, progression-free survival, and overall survival. In some embodiments, the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In some embodiments, the subject exhibits a progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody. In some embodiments, the subject exhibits an overall survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody.In some embodiments, the duration of response to the anti-CD70 antibody is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody. In some embodiments, the route of administration of the anti-CD70 antibody is intravenous. In some embodiments, the subject is human. In some embodiments, the anti-CD70 antibody is administered in combination with azacitidine. In some embodiments, the anti-CD70 antibody is administered in combination with venetoclax. In some embodiments, the anti-CD70 antibody is administered in combination with azacitidine and venetoclax. In some embodiments, the anti-CD70 antibody is administered in combination with a fluoroquinolone.

[0017] Also provided herein is a pharmaceutical composition for the treatment of a CD70-expressing cancer, comprising a nonfucosylated anti-CD70 antibody and at least one pharmaceutically compatible ingredient, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme, and wherein the pharmaceutical composition is for use in any of the methods of embodiments herein.

[0018] Also provided herein is a kit comprising a non-fucosylated anti-CD70 antibody, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme, and instructions for using the anti-CD70 antibody in any of the methods of the embodiments herein.

[0019] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0020] The patent or application file will contain at least one drawing executed in color. Copies of this patent or patent application publication and any color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows a series of sensograms of SGN-70 (fucosylated h1F6) and SEA-CD70 (nonfucosylated h1F6) binding to various Fcγ receptors. In Figure 1, SGN-70 is labeled as h1F6 WT, and SEA-CD70 is labeled as h1F6 SEA. Biolayer interferometry (BLI) was used to assess the binding kinetics and affinity of SGN-70 and SEA-CD70 to FcγR I, IIa, IIIa, IIb, and FcRN. [Figure 2] Figures 2A-2B show a series of graphs assessing the binding of SGN-70 and SEA-CD70 (labeled as SEA-70 in Figures 2A-2B) to the high-affinity human FcγRIIIa receptor (158V) (Figure 2A) or the cynomolgus monkey FcγRIIIa receptor (Figure 2B) using flow cytometry. [Figure 3] 3A-3B show a series of graphs depicting the ADCC activity of SGN-70 and SEA-CD70 in two CD70+ AML cell lines, MOLM-13 (FIG. 3A) and NOMO-1 (FIG. 3B). [Figure 4A-B]Figures 4A-4D show a series of graphs assessing the effects of SGN-70 (labeled as SGN-CD70 in Figures 4A-4D) and SEA-CD70 on CD70 Tregs and CD8 T cells in cells from donors homozygous for the high-affinity FcγRIIIa receptor (V / V 158) or homozygous for the low-affinity FcγRIIIa receptor (F / F 158). [Figure 4C-D] Figures 4A-4D show a series of graphs assessing the effects of SGN-70 (labeled as SGN-CD70 in Figures 4A-4D) and SEA-CD70 on CD70 Tregs and CD8 T cells in cells from donors homozygous for the high-affinity FcγRIIIa receptor (V / V 158) or homozygous for the low-affinity FcγRIIIa receptor (F / F 158). [Figure 4E-F] Figures 4E-4H show a series of graphs assessing the effect of fucosylated (WT clone 13 IgG1) or nonfucosylated (SEA clone 13 IgG1) anti-TIGIT antibodies on Tregs and CD8 T cells in cells from donors homozygous for the high-affinity FcγRIIIa receptor (V / V 158) or homozygous for the low-affinity FcγRIIIa receptor (F / F 158). [Figure 4G-H] Figures 4E-4H show a series of graphs assessing the effect of fucosylated (WT clone 13 IgG1) or nonfucosylated (SEA clone 13 IgG1) anti-TIGIT antibodies on Tregs and CD8 T cells in cells from donors homozygous for the high-affinity FcγRIIIa receptor (V / V 158) or homozygous for the low-affinity FcγRIIIa receptor (F / F 158). [Figure 5] 1 shows a Kaplan-Meyer graph assessing the effect of treatment with SEA-CD70 on animal survival over time in the Raji NHL Burkitt's lymphoma model. SEA-CD70 is labeled as h1F6SEA. [Figure 6]Graphs evaluating the antitumor effects of h1F6SEA, h1F6G1V1, h00SEA, and azacytidine in the MV-411 acute myeloid leukemia model are shown. SEA-CD70 is labeled as h1F6SEA. An antibody containing the same CDRs as SEA-CD70 but with inactivating backbone mutations is labeled as h1F6G1V1. A defucosylated human IgG1 isotype control antibody is labeled as h00SEA. [Figure 7A-B] Figures 7A-7D are a series of spider plots evaluating the antitumor effects of h1F6SEA, h00SEA (a defucosylated human IgG1 isotype control antibody), h1F6G1V1 (an antibody containing the same CDRs as SEA-CD70 but with inactivating backbone mutations), and azacytidine in the MV-411 acute myeloid leukemia model. Tumor volumes of individual animals are plotted for each treatment condition and overlaid with the median tumor volume of the untreated group. [Figure 7C-D] Figures 7A-7D are a series of spider plots evaluating the antitumor effects of h1F6SEA, h00SEA (a defucosylated human IgG1 isotype control antibody), h1F6G1V1 (an antibody containing the same CDRs as SEA-CD70 but with inactivating backbone mutations), and azacytidine in the MV-411 acute myeloid leukemia model. Tumor volumes of individual animals are plotted for each treatment condition and overlaid with the median tumor volume of the untreated group. [Figure 8] 8A-8B show a series of graphs assessing SEA-CD70 and SGN-CD70 mediated ADCP activity against AML cell lines. Data shown represent the percentage of positive macrophages relative to background control. [Figure 9] 9A-9B show a series of graphs assessing SEA-CD70 and SGN-CD70 CDC-mediated CDC activity against AML cell lines. [Figure 10]Graphs are shown evaluating the effect of SEA-CD70 in combination with azacitidine (VIDAZA®) on tumor growth in the MV411 AML xenograft mouse model. Mean tumor volumes (±SEM) are reported for each treatment arm. For each treatment group, data are plotted until the first animal in each group reaches a tumor size of over 1000 mm3. [Figure 11] Figures 11A-11B show a series of graphs evaluating the effect of SEA-CD70 in combination with azacitidine (VIDAZA®), venetoclax (VENCLEXTA®; ABT-199), or both (azacitidine + venetoclax) on tumor growth in the MV411 AML xenograft mouse model. Figure 11A: Mean tumor volume (±SEM) is reported for each treatment arm. For each treatment group, data are plotted until the first animal in each group reached a tumor size of >1000 mm3. Figure 11B: Single animal growth curves for control, azacitidine + venetoclax, and the SEA-CD70 + azacitidine + venetoclax combination (triplet combination). DETAILED DESCRIPTION OF THE INVENTION

[0022] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the described methods and compositions. When trade names are used herein, applicant intends to independently include the trade name product formulation, generic drug, and active pharmaceutical ingredient of the trade name product. As used herein, the following terms and phrases have the meanings ascribed to them unless otherwise specified.

[0023] As used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used herein in a phrase, e.g., "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in a phrase, e.g., "A, B, and / or C," is intended to encompass each of the following aspects: 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).

[0024] It will be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0025] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of this disclosure, but may have by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

[0026] The terms "CD70 binding agent" and "anti-CD70 binding agent," as used herein, refer to an anti-CD70 antibody, a derivative or fragment of an anti-CD70 antibody, or other agent that binds to CD70 and comprises at least one CDR or variable region of a CD70 binding antibody, or a derivative thereof.

[0027] The term "specifically binds" means that the binding agent reacts highly selectively with its corresponding antigen and does not react with many other antigens (eg, non-CD70 molecules).

[0028] As used herein, the term "functional" in the context of a CD70-binding agent indicates that the binding agent is capable of binding to CD70.

[0029] The terms "inhibit" or "inhibition," as used herein, mean to reduce by a measurable amount or to prevent completely.

[0030] The term "depletion" in the context of the effect of a CD70-binding agent on CD70-expressing cells refers to the reduction or elimination of the number of CD70-expressing cells.

[0031] "Intact antibody" and "intact immunoglobulin" are defined herein as heterotetrameric glycoproteins, typically about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by a disulfide bond to form a heterodimer. The heterotetramer is formed by covalent disulfide linkages between the two identical heavy chains of such a heterodimer. The light and heavy chains are linked together by disulfide bonds, although the number of disulfide linkages between the two heavy chains varies depending on the immunoglobulin (Ig) isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at its amino terminus. H ), followed by three or four constant domains (C H 1. C H 2. C H 3, and / or C H 4), and C H 1 and C H Each light chain has an amino-terminal variable domain (V L ) and a carboxy-terminal constant domain (C L ) has two domains. L Domain is V H noncovalently associated with the C domain L Domains are generally linked via disulfide bonds to C H Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., 1985, J. Mol. Biol. 186:651-663).

[0032] The term "hypervariable" refers to specific sequences within variable domains that differ significantly in sequence among antibodies and contain residues directly involved in the binding and specificity of each particular antibody for its specific antigenic determinant. Hypervariability in both the light-chain and heavy-chain variable domains is concentrated in three segments known as complementarity-determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, while HVLs are structurally defined according to the three-dimensional structure of the variable domains, as described in Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. While these two methods result in slightly different identifications of CDRs, the structural definition is preferred. As defined by Kabat (see Kabat et al., "Sequences of proteins of immunological interest," 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, MD, 1991), in the light chain variable domain, CDR-L1 is located at about residues 24-34, CDR-L2 is located at about residues 50-56, and CDR-L3 is located at about residues 89-97; in the heavy chain variable domain, CDR-H1 is located at about residues 31-35, CDR-H2 is located at about residues 50-65, and CDR-H3 is located at about residues 95-102.

[0033] The three CDRs in each heavy and light chain are separated by framework regions (FRs), which contain less variable sequences. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The primarily β-sheet structure of the FRs brings the CDRs within each chain into close proximity with each other and with the CDRs from the other chain. The resulting conformation contributes to the antigen-binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pp. 647-669), although not all CDR residues are directly involved in antigen binding.

[0034] Although FR residues and Ig constant domains are typically not directly involved in antigen binding, they can contribute to antigen binding or mediate antibody effector functions. Some FR residues can have a significant effect on antigen binding in at least three ways: by directly, non-covalently binding the epitope, by interacting with one or more CDR residues, and by influencing the interface between the heavy and light chains. The constant domains mediate antibody participation in various Ig effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0035] The light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa (k) and lambda (λ), based on the amino acid sequence of their constant domains. In contrast, the heavy chains of mammalian immunoglobulins are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM, according to the sequence of their constant domains. IgG and IgA are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

[0036] The terms "antibody," "anti-CD70 antibody," "humanized anti-CD70 antibody," and "variant humanized anti-CD70 antibody" are used herein in the broadest sense and specifically encompass full-length and native antibodies, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibodies or antigen-binding fragments thereof, e.g., variable domains and other portions of antibodies that exhibit the desired biological activity, e.g., CD70 binding.

[0037] The term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for any naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant, also called an epitope. The modifier "monoclonal" indicates a substantially homogeneous population of antibodies directed against the same epitope, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be produced by any technique or method known in the art, such as the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or by recombinant DNA methods known in the art (see, e.g., U.S. Pat. No. 4,816,567). In another example, monoclonal antibodies can be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352:624-628, and Marks et al., 1991, J. Mol. Biol. 222:581-597.

[0038] In contrast, the antibodies in a polyclonal antibody preparation are typically a heterogeneous population of immunoglobulin isotypes and / or classes, and display different epitope specificities.

[0039] The term "chimeric" antibody, as used herein, refers to a type of monoclonal antibody in which a partial or complete amino acid sequence in one or more regions or domains of the heavy and / or light chain is identical to, homologous to, or a variant of the corresponding sequence of a monoclonal antibody from another species or belonging to another immunoglobulin class or isotype, or derived from a consensus sequence. Chimeric antibodies include fragments of such antibodies, provided that they exhibit the desired biological activity of the parent antibody, e.g., bind to the same epitope (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al., 1984, Proc. Natl. Acad Sci. USA 81:6851-6855). Methods for producing chimeric antibodies are known in the art. (See, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397).

[0040] The terms "antibody fragment," "anti-CD70 antibody fragment," "humanized anti-CD70 antibody fragment," and "variant humanized anti-CD70 antibody fragment" refer to a portion of a full-length anti-CD70 antibody that retains the variable region or functional capability, e.g., specific CD70 epitope binding. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fd, Fv, scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies, and other multispecific antibodies formed from antibody fragments. (See Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.)

[0041] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V LA scFv polypeptide is optionally a single-chain Fv variant comprising a V domain, wherein the domain is present in a single polypeptide chain and is capable of recognizing and binding to an antigen. H Domains and V L The scFv contains a polypeptide linker positioned between the domains, which enables the scFv to form the desired three-dimensional structure for antigen binding (see, e.g., Pluckthun, 1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315).

[0042] The term "diabody" refers to a small antibody fragment with two antigen-binding sites. Each fragment contains a light chain variable domain (V L ) linked to a heavy chain variable domain (V H ) and V H -V L or V L -V H By using a linker that is too short to allow pairing between the two domains on the same chain, the linked V H -V L The domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in further detail in, for example, EP 404097; WO 93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0043] The term "linear antibody" refers to an antibody that comprises a pair of tandem Fd segments (V) that form a pair of antigen-binding regions. H -C H 1-V H -C H 1). Linear antibodies can be bispecific or monospecific, as described in Zapata et al., 1995, Protein Eng. 8(10):1057-1062.

[0044] A "humanized antibody" refers to an immunoglobulin amino acid sequence variant or fragment thereof that is capable of binding to a predetermined antigen and comprises variable region polypeptide chains having framework regions substantially having the amino acid sequence of a human immunoglobulin and CDRs substantially having the amino acid sequence of a non-human immunoglobulin.

[0045] Generally, humanized antibodies have one or more amino acid residues introduced into them from a source that is non-human. These non-human amino acid residues, referred to herein as "import" residues, are typically taken from an "import" antibody domain, particularly a variable domain. The import residues, sequence, or antibody possess the desired affinity and / or specificity or other desired antibody biological activity, as discussed herein.

[0046] Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are derived from human immunoglobulin sequences, e.g., consensus or germline sequences. The humanized antibody also optionally comprises at least a portion of an immunoglobulin Fc domain, typically that of a human immunoglobulin. For example, the antibody can comprise both a light chain and at least the variable domain of a heavy chain. The antibody also optionally comprises the C of the heavy chain. H 1. Hinge (J), C H 2. C H 3, and / or C H It may contain four regions.

[0047] Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. The constant region or domain can include, for example, a complement-fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity (e.g., IgG1). If such cytotoxic activity is not desired, the constant domain can be of another class (e.g., IgG2). Humanized antibodies can contain sequences from more than one class or isotype, and it is within the skill of the art to select a particular constant domain to optimize desired effector functions.

[0048] The FR and CDR regions of a humanized antibody need not correspond exactly to those of the parental sequences; for example, the import CDR or consensus FR may be altered by substitution, insertion, or deletion of at least one residue so that the CDR or FR residue at that site does not correspond to either the consensus or import antibody. Such mutations are typically not extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences, more often at least 90%, and most often more than 95%.

[0049] The term "antibody effector function," as used herein, refers to a function contributed by the Fc domain of an Ig. Such a function can be, for example, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, or complement-dependent cytotoxicity. Such a function can be mediated, for example, by binding of the Fc effector domain to an Fc receptor on an immune cell with phagocytic or lytic activity, or by binding of the Fc effector domain to a component of the complement system. Typically, effects mediated by Fc-binding cells or complement components result in the inhibition and / or depletion of CD70 target cells. Without intending to be bound by any particular theory, the Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and juxtapose them to antibody-coated target cells. Cells expressing surface FcRs for IgG, including FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64), can act as effector cells for the destruction of IgG-coated cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils. Engagement of FcγR by IgG activates antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). ADCC involves the activation of CD16 through the secretion of pore-forming proteins and proteases. + Phagocytosis is mediated by effector cells, and CD32 + and CD64 +It is mediated by effector cells (see Fundamental Immunology, 4th ed., Paul, ed., Lippincott-Raven, NY, 1997, Chapters 3, 17, and 30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207). In addition to ADCC and ADCP, the Fc region of cell-bound antibodies can also activate the classical complement pathway to induce complement-dependent cytotoxicity (CDC). C1q of the complement system binds to the Fc region of antibodies when complexed with antigen. Binding of C1q to cell-bound antibodies can initiate a cascade of events involving the proteolytic activation of C4 and C2 to generate C3 convertase. Cleavage of C3 to C3b by C3 convertase allows activation of terminal complement components including C5b, C6, C7, C8, and C9. Collectively, these proteins form membrane attack complex pores on antibody-coated cells. These pores disrupt the integrity of the cell membrane and kill the target cell (see Immunobiology, 6th ed., Janeway et al., Garland Science, NY, 2005, Chapter 2).

[0050] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism for inducing cell death that relies on the interaction of antibody-coated target cells with immune cells (also called effector cells) that have lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells bind to the Fc effector domain of Ig bound to target cells via the antigen-binding site. Death of antibody-coated target cells occurs as a result of the activity of effector cells.

[0051] The term "antibody-dependent cellular phagocytosis" or ADCP refers to the process by which antibody-coated cells are ingested, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain of Ig.

[0052] The term "complement-dependent cytotoxicity" or CDC refers to a cell death-inducing mechanism in which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that result in the formation of holes in the target cell membrane. Typically, antigen-antibody complexes, e.g., on antibody-coated target cells, bind and activate complement component C1q, which in turn activates the complement cascade that leads to target cell death. Complement activation can also result in the deposition of complement components on the target cell surface that promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0053] "Immune cell," as used herein, refers to a cell of the hematopoietic lineage that is involved in regulating the immune response. In typical embodiments, the immune cell is a T lymphocyte, a B lymphocyte, a NK cell, a monocyte / macrophage, or a dendritic cell.

[0054] As used herein, the term "effector cell" refers to a cell that expresses a surface receptor for the Fc domain of immunoglobulin (FcR). For example, cells that express surface FcRs for IgG, including FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64), can act as effector cells. Such effector cells include monocytes, macrophages, natural killer (NK) cells, neutrophils, and eosinophils.

[0055] A "therapeutic agent" is an agent that exerts a cytotoxic, cytostatic, and / or immunomodulatory effect on cancer cells, activated immune cells, or other target cell populations. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.

[0056] "Cytotoxic effect" refers to the depletion, elimination, and / or killing of target cells. "Cytotoxic agent" refers to an agent that has a cytotoxic effect on cells. The term includes radioisotopes (e.g., I 131 , I 125 , Y 90 , and Re 186 ), chemotherapeutic agents, and toxins, e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof. Such cytotoxic agents can be coupled to an antibody, e.g., a humanized anti-CD70 antibody, to treat, for example, a patient indicated for antibody therapy. In one embodiment, a "cytotoxic agent" includes a monoclonal antibody, e.g., an antibody used in combination with a humanized antibody described herein.

[0057] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs) and its derivatives; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins, auristatins (including the analogs monomethyl-auristatin E and monomethyl-auristatin B). (including benzodiazepine F) (see, for example, U.S. Patent Application Publication No. 2005-0238649, published October 27, 2005, which is incorporated herein in its entirety); duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, iphosphatidylcholine, sulfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, novembitine, phenesterine, prednimustine; trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., endene antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin phiI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186)); dynemicins, e.g., dynemicin A; bisphosphonates, e.g., clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin™), (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogues, e.g., denopterin, methotrexate, pteropterin, tocopherol, thiazolinone, thiazolinone, thiazolinone, thiazolinone; Rimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadranals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; Aceglatone; Aldophosphamide glucoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Democorsin; Diaziquone; Eflornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone, mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarbucin;losoxantrone, podophyllic acid; 2-ethylhydrazine; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitabronitol; mitrachlor; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action in tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™);Aromatase inhibitors, which inhibit aromatase, an enzyme that controls estrogen production in the adrenal glands, include, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0058] The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance, which is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into a more active parent form.See, for example, Wilman, 1986, "Prodrugs in Cancer Chemotherapy", In Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast; and Stella et al., 1985, Prodrugs: A Chemical Approach to Targeted Drug Delivery, In: Directed Drug Delivery, Borchardt et al. (eds.), pp. 247-267, Humana Press. Useful prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active, non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms include, but are not limited to, those chemotherapeutic agents described above.

[0059] A "cytostatic effect" refers to the inhibition of cell proliferation. A "cytostatic agent" refers to an agent that has a cytostatic effect on cells, thereby inhibiting the proliferation and / or expansion of specific subsets of cells.

[0060] As used herein, the term "immunomodulatory effect" refers to stimulating (immunostimulating) or inhibiting (immunosuppressing) the development or maintenance of an immunological response. Inhibition can be achieved, for example, by eliminating immune cells (e.g., T or B lymphocytes); inducing or generating immune cells that can modulate (e.g., downregulate) the functional capacity of other cells; inducing a non-responsive state in immune cells (e.g., anergy); or increasing, decreasing, or altering the activity or function of immune cells, for example, by changing the pattern of proteins expressed by the immune cell (e.g., altered production and / or secretion of certain molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors). An "immunomodulatory agent" refers to an agent that has an immunomodulatory effect on cells. In some embodiments, an immunomodulatory agent has a cytotoxic or cytostatic effect on immune cells that promotes an immune response.

[0061] The term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labeled anti-CD70 antibodies can be prepared and used in a variety of applications, including in vitro and in vivo diagnostics.

[0062] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that normally express an antibody, for example, when the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0063] The term "control sequence" refers to polynucleotide sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for use in prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences. Eukaryotic control sequences include, but are not limited to, promoters, polyadenylation signals, and enhancers. These control sequences can be utilized for the expression and production of anti-CD70 binding agents in prokaryotic and eukaryotic host cells.

[0064] A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers are optionally contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used to join the DNA sequences.

[0065] The term "polypeptide" refers to a polymer of amino acids or its equivalent, and does not refer to a specific length of the product; therefore, "peptide" and "protein" are included in the definition of polypeptide. Also included in the definition of polypeptide is an "antibody," as defined herein. A "polypeptide region" refers to a segment of a polypeptide, which may include, for example, one or more domains or motifs (e.g., a polypeptide region of an antibody may include, for example, one or more complementarity-determining regions (CDRs)). The term "fragment" typically refers to a portion of a polypeptide having at least 20 contiguous or at least 50 contiguous amino acids of the polypeptide. A "derivative" is a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid substitutions with respect to a second polypeptide; or a polypeptide or fragment thereof that has been altered (modified), for example, by conjugation of a heterologous polypeptide or by the covalent attachment of a second molecule, such as by glycosylation, acetylation, phosphorylation, etc. Also included within the definition of "derivative" are, for example, polypeptides containing one or more analogs of an amino acid (such as, for example, unnatural amino acids), polypeptides with unsubstituted linkages, as well as other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0066] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated polypeptides include isolated antibodies, or fragments or derivatives thereof. "Antibody" includes antibodies in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.

[0067] In certain embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and in other aspects to greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.

[0068] The term "heterologous" in the context of a polypeptide means derived from a different source (e.g., a cell, tissue, organism, or species) compared to another polypeptide such that the two polypeptides are different. Typically, a heterologous polypeptide is derived from a different species.

[0069] In the context of an immunoglobulin polypeptide or a fragment thereof, a "conservative substitution" refers to a substitution that maintains specific binding of the immunoglobulin polypeptide or a fragment thereof to an antigen (e.g., K D "Affinity" refers to one or more amino acid substitutions that do not substantially reduce binding affinity (i.e., substitutions that increase binding affinity, do not significantly alter binding affinity, or reduce binding affinity by no more than about 40%, typically no more than about 30%, more typically no more than about 20%, even more typically no more than about 10%, or most typically no more than about 5% as determined by a standard binding assay, such as ELISA).

[0070] The terms "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, are identical or have a specified percentage of identical nucleotide or amino acid residues. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences are of identical length.

[0071] In the context of two nucleic acids or polypeptides, the term "substantially identical" refers to two or more sequences or subsequences that have at least 50%, at least 55%, at least 60%, or at least 65% identity, typically at least 70% or at least 75% identity, more typically at least 80% or at least 85% identity, and even more typically at least 90%, at least 95%, or at least 98% identity (e.g., as determined using one of the methods set out below).

[0072] The term "similarity" or "percent similarity," in the context of two or more polypeptide sequences, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have a specified percentage of amino acid residues that are identical or conservatively substituted, as measured using one of the methods set forth below. By way of example, a first amino acid sequence can be considered similar to a second amino acid sequence if it is at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical to or conservatively substituted with the second amino acid sequence when compared over the same number of amino acids as contained in the first sequence, or, for example, when compared against an alignment of polypeptides aligned by one of the methods set forth below.

[0073] In the context of polypeptide sequences, the terms "substantial similarity" or "substantially similar" indicate that a polypeptide region has a sequence having at least 70%, typically at least 80%, and more typically at least 85% or at least 90% or at least 95% sequence similarity to a reference sequence. For example, a polypeptide is substantially similar to a second polypeptide when, for example, the two peptides differ by one or more conservative substitutions.

[0074] In the context of an anti-CD70 antibody or derivative thereof, a protein having one or more polypeptide regions substantially identical to or substantially similar to one or more antigen-binding regions of an anti-CD70 antibody (e.g., a heavy or light chain variable region or a heavy or light chain CDR) retains specific binding to the epitope of CD70 recognized by the anti-CD70 antibody, as determined using any of a variety of standard immunoassays known in the art or as referred to herein.

[0075] The determination of percent identity or similarity between two sequences can be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87, 2264-2268, modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90, 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215, 403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25, 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table of 12, a gap length penalty of 12, and a gap penalty of 4 can be used.Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10, 3-5, and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, regions of similarity in the two sequences being compared are found by examining aligned residue pairs; when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1-6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments may be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266, 383-402.

[0076] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or naturally occurring mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are also included. Where separate designations are intended, it will be clear from the context.

[0077] The term "subject" for purposes of treatment refers to any animal, particularly an animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the subject is a human.

[0078] As used herein, the terms "disorder" and "CD70-associated disorder" and "CD70-associated disease" refer to any condition that would benefit from treatment with an anti-CD70 binding agent as described herein. "CD70-associated disorder" and "CD70-associated disease" generally refer to CD70 or a fragment thereof on the cell surface. This includes chronic and acute disorders or diseases, including those conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer, myeloid malignancies, hematological malignancies, benign and malignant tumors, leukemia and lymphoid malignancies, carcinomas, and inflammatory, angiogenic, and immunological disorders. Specific examples of disorders are disclosed below.

[0079] As used herein, terms such as "treatment" and "therapy" are meant to include therapeutic as well as prophylactic or preventative measures for a disease or disorder that result in any clinically desired or beneficial effect, including, but not limited to, the alleviation or alleviation of one or more symptoms, the regression of a disease or disorder, or the slowing or halting of its progression. Thus, for example, the term treatment includes the administration of an agent before or after the onset of symptoms of a disease or disorder, thereby preventing or eliminating all signs of the disease or disorder. As another example, the term includes the administration of an agent after clinical manifestation of a disease to combat disease symptoms. Furthermore, the administration of an agent after onset and after the occurrence of clinical symptoms includes "treatment" or "therapy" as used herein, regardless of whether the treatment results in disease remission, if the administration affects clinical parameters of the disease or disorder, such as the degree of tissue damage or the amount or extent of metastasis.

[0080] As used herein, the terms "prevention" or "preventing" refer to administration of an anti-CD70 binding agent to a subject prior to the onset of clinical or diagnostic symptoms of a CD70-expressing cancer or immunological disorder (e.g., administration to an individual who is predisposed to or at high risk of acquiring a CD70-expressing cancer or immunological disorder) to (a) block the occurrence or development of a CD70-expressing cancer or immunological disorder, or one or more of its clinical or diagnostic symptoms; (b) inhibit the severity of the onset of a CD70-expressing cancer or immunological disorder; or (c) reduce the likelihood of developing a CD70-expressing cancer or immunological disorder.

[0081] The term "intravenous infusion" refers to the introduction of a drug, eg, a therapeutic agent, into the vein of an animal or human patient over a period of more than approximately 15 minutes, generally approximately 30 to 90 minutes.

[0082] The term "intravenous bolus" or "intravenous push" refers to the administration of a drug into the vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, generally 5 minutes or less.

[0083] The term "subcutaneous administration" refers to the introduction of a drug, e.g., a therapeutic agent, under the skin of an animal or human patient, usually into a pocket between the skin and the underlying tissue, by relatively slow, sustained delivery from a drug reservoir. The pocket may be created by pinching or pulling the skin away from the underlying tissue.

[0084] The term "package insert" is used to refer to instructions customarily included in the packaging of commercially available therapeutic products, which contain information regarding the indications, usage, administration, contraindications and / or warnings regarding the use of such therapeutic products.

[0085] "Liposomes" are small vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for delivery of drugs (e.g., antibodies) to mammals. The components of a liposome are usually arranged in a bilayer structure, similar to the lipid arrangement of biological membranes.

[0086] The term "subcutaneous infusion" refers to the introduction of a drug under the skin of an animal or human patient, preferably into the pocket between the skin and the underlying tissue, by relatively slow, sustained delivery from a drug reservoir for a period including, but not limited to, 30 minutes or less or 90 minutes or less. Optionally, the infusion may be performed by subcutaneous implantation of a drug delivery pump that is implanted under the skin of the animal or human patient, the pump delivering a predetermined amount of drug for a predetermined period of time, e.g., 30 minutes, 90 minutes, or the length of a treatment regimen.

[0087] The term "subcutaneous bolus" refers to drug administration just beneath the skin of an animal or human patient, with the bolus drug delivery taking less than approximately 15 minutes, in another embodiment less than 5 minutes, and in yet another embodiment less than 60 seconds. In still yet another embodiment, administration is within a pocket between the skin and the underlying tissue, which may be created by pinching or pulling the skin away from the underlying tissue.

[0088] The term "effective amount" refers to an amount of an anti-CD70 binding agent (e.g., an antibody or derivative or other binding agent) sufficient to inhibit the development of or ameliorate one or more clinical or diagnostic symptoms of a CD70-expressing cancer or immunological disorder in a subject. An effective amount of the agent is administered according to the methods described herein in an "effective regimen." The term "effective regimen" refers to a combination of the amount of agent and frequency of administration appropriate to achieve treatment or prevention of a CD70-expressing cancer or immunological disorder.

[0089] The term "therapeutically effective amount" is used to refer to an amount of a therapeutic agent that has a beneficial patient outcome, e.g., a growth-arresting effect or deletion of cells. In one aspect, a therapeutically effective amount has apoptotic activity or is capable of inducing cell death. In another aspect, a therapeutically effective amount refers to a target serum concentration that has been shown to be effective, e.g., in slowing disease progression. Efficacy can be measured by conventional methods depending on the condition to be treated. For example, in neoplastic diseases or disorders characterized by cells expressing CD70, efficacy can be measured by assessing the time to disease progression (TTP) or by determining the response rate (RR).

[0090] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameters (SLD) of the target lesions, referenced to the baseline SLD, and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify for PR nor sufficient growth to qualify for PD, referenced to the smallest SLD since treatment began.

[0091] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete or partial response as well as the amount of time a patient experiences stable disease.

[0092] As used herein, "overall response rate" or "ORR" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0093] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to be alive after a particular period of time.

[0094] An "adverse event" (AE), as used herein, is any untoward, generally unintended, or undesirable sign (including abnormal laboratory findings), symptom, or disease associated with the use of a medical treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. Reference to methods that can "modify adverse events" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0095] A "serious adverse event" or "SAE," as used herein, is an adverse event that meets one of the following criteria: Fatal or life-threatening ("life-threatening," as used in the definition of serious adverse events, refers to an event in which the patient was at risk of death at the time of the event, not an event that, if more severe, might hypothetically have caused death) ·Causes permanent or significant disability / incapacity · Congenital anomalies / congenital defects Medically significant, i.e., defined as an event that may endanger the patient or require medical or surgical intervention to prevent one of the outcomes listed above. Medical and scientific judgment must be exercised in determining whether an AE is "medically significant." Requiring inpatient hospitalization or an extension of an existing hospitalization, except for: 1) routine treatment or monitoring of an underlying disease that is not associated with any worsening of the condition, 2) elective or pre-planned treatment for a pre-existing condition unrelated to the indication under study that has not worsened since signing the informed consent, and 3) social reasons and respite care in the absence of any worsening of the patient's general condition.

[0096] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or enumerated components.

[0097] The terms "about" or "essentially consisting of" refer to a value or composition that is within an acceptable error range of the particular value or composition as determined by one of ordinary skill in the art, which will vary somewhat depending on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within 1 or more standard deviations per practice in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 20%. Moreover, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range of that particular value or composition.

[0098] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or vehicle with which an anti-CD70 binding agent is administered.

[0099] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of an anti-CD70 binding agent or therapeutic agent. The anti-CD70 binding agent or therapeutic agent contains at least one amino group and, therefore, acid addition salts can be formed with this amino group or other suitable group. 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, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. Counter ions can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts can have atoms with two or more charges in their structure. In the case where multiple charged atoms are part of a pharmaceutically acceptable salt, they may have multiple counter ions. Therefore, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counter ions.

[0100] A "pharmaceutically acceptable solvate" or "solvate" refers to an association of one or more solvent molecules and an anti-CD70 binding agent and / or therapeutic agent. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0101] The abbreviation "AFP" refers to dimethylvaline-valine-dolaisoloiin-dolaproine-phenylalanine-p-phenylenediamine.

[0102] The abbreviation "MMAE" refers to monomethyl auristatin E.

[0103] The abbreviation "AEB" refers to the ester produced by reacting auristatin E with paraacetylbenzoic acid.

[0104] The abbreviation "AEVB" refers to the ester produced by reacting auristatin E with benzoylvaleric acid.

[0105] The abbreviation "MMAF" refers to dovaline-valine-dolaisoleunine-dolaproine-phenylalanine.

[0106] The abbreviations "fk" and "phe-lys" refer to the linker phenylalanine-lysine.

[0107] "Treg" or "regulatory T cells" are CD4+CD25 + and CD8 + CD4 suppresses T cell proliferation and / or effector function or otherwise down-regulates the immune response + Refers to T cells. Notably, Tregs can downregulate immune responses mediated by natural killer cells, natural killer T cells, and other immune cells.

[0108] The term "regulatory T cell function" or "Treg function" refers to CD4+CD25 + or CD8 + The terms "Treg" and "Treg" are used interchangeably to refer to any biological function of Tregs that results in reduced T cell proliferation or reduced effector T cell-mediated immune responses. Treg function can be measured by techniques established in the art. Non-limiting examples of useful in vitro assays for measuring Treg function include the Transwell suppression assay and assays in which target conventional T cells (Tconv) and Tregs purified from human peripheral blood or umbilical cord blood (or mouse spleen or lymph nodes) are stimulated with anti-CD3 +Conventional in vitro assays include those in which T cell proliferation is detected in vitro (e.g., by measuring incorporation of radioactive nucleotides (e.g., [H]-thymidine) or fluorescent nucleotides, or by the Cayman Chemical MTT cell proliferation assay kit, or by monitoring the dilution of green fluorescent ester CFSE or seminaphtharhodafluor (SNARF-1) dyes by flow cytometry), followed by optional activation with anti-CD28 coated beads (or antigen-presenting cells (APCs), such as irradiated splenocytes or purified dendritic cells (DCs), or irradiated PBMCs). Other common assays measure T cell cytokine responses. Useful in vivo assays of Treg function include assays in animal models of diseases in which Tregs play a key role, such as (1) homeostatic models (naive, homeostatically expanding CD4 T cells as target cells primarily suppressed by Tregs) + (2) an inflammatory bowel disease (IBD) recovery model (using Thl T cells (Thl7) as target cells primarily suppressed by Tregs); (3) an experimental autoimmune encephalomyelitis (EAE) model (using Thl7 and Thl T cells as target cells primarily suppressed by Tregs); (4) a B16 melanoma model (suppression of antitumor immunity) (using CD8 T cells as target cells primarily suppressed by Tregs). + T cells), (5) naive CD4 + CD45RB M These include suppression of colonic inflammation in adoptive transfer colitis, in which Tconv cells are transplanted into RagV mice, and (6) the Foxp3 rescue model, which uses lymphocytes as target cells that are primarily suppressed by Tregs. According to one protocol, all of the models involve mice for the donor T cell population and Ragl for the recipient. - / -For more details on various useful assays, see, for example, Collison and Vignali, In vitro Treg Suppression Assays, Chapter 2: Methods and Protocols in Regulatory T Cells, Methods in Molecular Biology, Kassiotis and Liston (eds.), Springer, 2011, 707:21-37; Workman et al., In vivo Treg Suppression Assays, Chapter 9: Methods and Protocols in Regulatory T Cells, Methods in Molecular Biology, Kassiotis and Liston (eds.), Springer, 2011, 119-156; Takahashi et al., Int. Immunol., 1998, 10:1969-1980; Thornton et al., J. Exp. Med., 1998, 188:287-296; Collison et al., J. Immunol, 2009, 182:6121-6128; Thornton and Shevach, J. Exp. Med., 1998, 188:287-296; Asseman et al., J. Exp. Med., 1999, 190:995-1004; Dieckmann et al., J. Exp. Med., 2001, 193:1303-1310; Belkaid, Nature Reviews, 2007, 7:875-888; Tang and Bluestone, Nature Immunology, 2008, 9:239-244; Bettini and Vignali, Curr. Opin. Immunol, 2009, 21:612-618; Dannull et al., J. Clin See Invest, 2005, 115(12):3623-33; Tsaknaridis, et al., J Neurosci Res., 2003, 74:296-308.

[0109] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless otherwise specified.

[0110] Various aspects of the disclosure are described in further detail in the following subsections.

[0111] II. Anti-CD70 antibody The present invention provides anti-CD70 antibodies, such as humanized antibodies derived from the murine antibody 1F6. 1F6 is a murine immunoglobulin G1 (IgG1) monoclonal antibody against CD70. 1F6 and humanized 1F6 variants are described in U.S. Pat. No. 8,067,546 and International Patent Publication WO 2006 / 113909. In some embodiments, the anti-CD70 antibody is non-fucosylated.

[0112] The binding affinity (i.e., dissociation constant, K) of the humanized form of the murine 1F6 antibody D ) is preferably within 5-fold or 2-fold of that of the murine antibody 1F6 for human CD70. Humanized 1F6 antibodies, like the murine antibody from which they are derived, specifically bind to human CD70 in its native form and / or recombinantly expressed from Chinese hamster ovary (CHO) cells. Preferred humanized 1F6 antibodies have an affinity for human CD70 that is the same as or greater than that of 1F6 (i.e., greater than the acceptable range of error in measurement) (e.g., 1.1-5 times, 1.1-3 times, 1.5-3 times, 1.7-2.3 times, or 1.7-2.1 times the affinity of 1F6, or about 2 times). Preferred humanized 1F6 antibodies bind to the same epitope as 1F6 for binding to human CD70 and / or compete with 1F6 for binding to human CD70.

[0113] In some embodiments, antibodies of the invention inhibit cancer (e.g., cell proliferation, metastasis, and / or organ lethality) as demonstrated in cancerous cells grown in culture, in animal models, or in clinical trials. Animal models can be generated by implanting CD70-expressing human tumor cell lines into appropriate immunodeficient rodent strains, such as athymic nude mice or SCID mice. These tumor cell lines can be established in the immunodeficient rodent host as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection.

[0114] Once established in the host, these tumor models can be applied to evaluate the therapeutic efficacy of anti-CD70 antibodies or conjugated forms thereof, as described in the Examples.

[0115] Generally, anti-CD70 antibodies of the present disclosure bind to CD70, e.g., human CD70, and exert cytostatic and cytotoxic effects on malignant cells, e.g., cancer cells. Anti-CD70 antibodies of the present disclosure are preferably monoclonal and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, and CD70-binding fragments of any of the above. In some embodiments, anti-CD70 antibodies of the present disclosure specifically bind to CD70. Immunoglobulin molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0116] In certain embodiments of the present disclosure, the anti-CD70 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) as described herein, including, but not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), and V L or V HAntigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. Also included in the disclosure are antigen-binding fragments comprising any combination of the variable region(s) with the hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-CD70 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.

[0117] The anti-CD70 antibodies of the present disclosure may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of CD70, or may be specific for both CD70 and a heterologous protein. See, e.g., PCT Publications WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, WO 92 / 05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[0118] The anti-CD70 antibody of the present disclosure may be a humanized antibody. In some embodiments, the anti-CD70 antibody of the present disclosure is a humanized version of the murine antibody 1F6. A humanized version of 1F6 is described in U.S. Patent No. 8,067,546. A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see, e.g., Queen, U.S. Patents 5,530,101 and 5,585,089; Winter, U.S. Patent 5,225,539; Carter, U.S. Patent 6,407,213; Adair, U.S. Patent 5,859,205; and Foote, U.S. Patent 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. A preferred acceptor sequence for the heavy chain is the germline V H Exon V H l-2 (also called HV1-2 in the literature) (Shin et al., 1991, EMBO J. 10:3641-3645), and the hinge region (J H ) for Exon J H-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30), and for the hinge region, exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is one having some or all of its CDRs completely or substantially derived from a donor antibody, and variable region framework sequences and constant regions, if present, completely or substantially derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody heavy chain, and heavy chain variable region framework sequences and heavy chain constant regions, if present, substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially derived from human light chain variable region framework and constant region sequences. Humanized antibodies, other than nanobodies and dAbs, comprise a humanized heavy chain and a humanized light chain. CDRs in a humanized antibody are substantially derived from corresponding CDRs in a non-human antibody if at least 60%, 85%, 90%, 95%, or 100% (as defined by Kabat) of corresponding residues are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially derived from human variable region framework sequences or human constant regions, respectively, if at least 85%, 90%, 95%, or 100% of corresponding residues as defined by Kabat are identical.

[0119] Humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a murine antibody, although they can also be made with fewer than all CDRs from a murine antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0120] Certain amino acids from the human variable region framework residues may be selected for substitution based on their potential effect on CDR conformation and / or binding to antigen, with investigation of such potential effect being by modeling, examination of the characteristics of the amino acid at a particular position, or experimental observation of the effects of substitution or mutagenesis of a particular amino acid.

[0121] For example, if an amino acid differs between the murine variable region framework residue and the selected human variable region framework residue, the human framework amino acid may be selected such that the amino acid is: (1) Directly binds to the antigen noncovalently; (2) adjacent to the CDR region, (3) otherwise interacts with the CDR region (e.g., is within about 6 A of the CDR region), or (4) Mediates the interaction between heavy and light chains The amino acids may be substituted with the equivalent framework amino acids from the mouse antibody if it is reasonably expected that they will be.

[0122] The anti-CD70 antibodies of the present disclosure may be described or designated in terms of the particular CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be determined by the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme), Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jul;309(3):657-70, ("Aho" numbering scheme), and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm", PNAS, 1989,86(23):9268-9272, ("AbM" numbering scheme).The boundaries of a given CDR may vary depending on the scheme used for its identification. In some embodiments, the "CDR" or "complementarity determining region" or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) should be understood to encompass the CDRs as defined (or designated) by any of the above schemes. For example, if a particular CDR (e.g., CDR-H3) is located within a given V H or V L When a region amino acid sequence is described as containing the amino acid sequence of a corresponding CDR in the variable region, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region as defined by any of the above schemes. Schemes for identifying specific CDR(s) can be specified, such as CDRs as defined by the Kabat, Chothia, AbM, or IMGT methods.

[0123] The CDR sequences of the anti-CD70 antibodies and of the anti-CD70 antibody-drug conjugates described herein follow the Kabat numbering scheme as described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, unless otherwise specified.

[0124] In one aspect, provided herein is an anti-CD70 antibody comprising a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1 and a light chain variable region comprising the three CDRs of SEQ ID NO: 2, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme. In some embodiments, the anti-CD70 antibody further comprises an Fc domain. In some embodiments, the anti-CD70 antibody is non-fucosylated.

[0125] The anti-CD70 antibodies described herein can comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind to CD70 (e.g., human CD70). As used herein, heavy chain framework regions are designated as "HC-FR1-FR4" and light chain framework regions are designated as "LC-FR1-FR4."

[0126] In some embodiments of the anti-CD70 antibodies described herein, the heavy chain variable domain comprises: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS (SEQ ID NO: 1), and the light chain variable domain comprises the amino acid sequence of It contains the amino acid sequence of DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLES GVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK (SEQ ID NO: 2).

[0127] In some embodiments of the anti-CD70 antibodies described herein, the heavy chain variable domain comprises: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS (SEQ ID NO: 1), and the light chain variable domain comprises the amino acid sequence of It contains the amino acid sequence of DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLES GVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIKR (SEQ ID NO: 7).

[0128] In one aspect, provided herein is an anti-CD70 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, or comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. In one aspect, provided herein is an anti-CD70 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0129] In one aspect, provided herein is an anti-CD70 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, or comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. In one aspect, provided herein is an anti-CD70 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0130] In some embodiments, provided herein are anti-CD70 antibodies comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. In certain embodiments, an amino acid sequence heavy chain variable domain having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to CD70 (e.g., human CD70). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 1. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, an anti-CD70 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 1, including post-translational modifications of that sequence. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0131] In some embodiments, provided herein are anti-CD70 antibodies comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to CD70 (e.g., human CD70). In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 2 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-CD70 antibody comprises the light chain variable domain sequence of SEQ ID NO: 2, including post-translational modifications of that sequence.

[0132] In some embodiments, provided herein are anti-CD70 antibodies comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to CD70 (e.g., human CD70). In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 7 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-CD70 antibody comprises the light chain variable domain sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

[0133] In some embodiments, the amino acid sequence QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLKWMGW INTYTGEPTY ADAFKGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARDY GDYGMDYWGQ GTTVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV Provided herein are anti-CD70 antibodies comprising a heavy chain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK (SEQ ID NO: 3). In certain embodiments, a heavy chain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to CD70 (e.g., human CD70). In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 3 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, an anti-CD70 antibody comprises the heavy chain sequence of SEQ ID NO: 3, including post-translational modifications of that sequence.

[0134] In some embodiments, provided herein are anti-CD70 antibodies comprising a light chain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of DIVMTQSPDS LAVSLGERAT INCRASKSVS TSGYSFMHWY QQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSREVPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 4). In certain embodiments, a light chain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to CD70 (e.g., human CD70). In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 4 have been substituted, inserted, and / or deleted. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, an anti-CD70 antibody comprises the light chain sequence of SEQ ID NO: 4, including post-translational modifications of that sequence.

[0135] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable domain as in any of the embodiments provided above and a light chain variable domain as in any of the embodiments provided above. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 1 and the light chain variable domain sequence of SEQ ID NO: 2, including post-translational modifications of those sequences. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0136] In some embodiments, the anti-CD70 antibody comprises i) an amino acid sequence having at least 85% sequence identity to a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and ii) an amino acid sequence having at least 85% sequence identity to a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0137] In some embodiments, the anti-CD70 antibody is a monoclonal antibody.

[0138] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising three CDRs or a light chain variable region comprising three CDRs of an anti-CD70 antibody described in U.S. Pat. No. 8,067,546, U.S. Pat. No. 8,562,987, U.S. Pat. No. 9,428,585, U.S. Pat. No. 9,701,752, US 2009 / 0148942, US 2012 / 0045436, US 2014 / 0178936, US 2017 / 0022282, or International Patent Publication WO 2006 / 113909. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising three CDRs and a light chain variable region comprising three CDRs of an anti-CD70 antibody described in U.S. Patent No. 8,067,546, U.S. Patent No. 8,562,987, U.S. Patent No. 9,428,585, U.S. Patent No. 9,701,752, US 2009 / 0148942, US 2012 / 0045436, US 2014 / 0178936, US 2017 / 0022282, or International Patent Publication WO 2006 / 113909. In some embodiments, the CDRs are defined by the Kabat numbering scheme.

[0139] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region or a light chain variable region of an anti-CD70 antibody described in U.S. Pat. No. 8,067,546, U.S. Pat. No. 8,562,987, U.S. Pat. No. 9,428,585, U.S. Pat. No. 9,701,752, US 2009 / 0148942, US 2012 / 0045436, US 2014 / 0178936, US 2017 / 0022282, or International Patent Publication WO 2006 / 113909. In some embodiments, the anti-CD70 antibody comprises the heavy chain variable region and the light chain variable region of an anti-CD70 antibody described in U.S. Pat. No. 8,067,546, U.S. Pat. No. 8,562,987, U.S. Pat. No. 9,428,585, U.S. Pat. No. 9,701,752, US 2009 / 0148942, US 2012 / 0045436, US 2014 / 0178936, US 2017 / 0022282, or International Patent Publication WO 2006 / 113909.

[0140] In some embodiments, the anti-CD70 antibody is an anti-CD70 antibody such as those described in U.S. Pat. No. 8,067,546, U.S. Pat. No. 8,562,987, U.S. Pat. No. 9,428,585, U.S. Pat. No. 9,701,752, US 2009 / 0148942, US 2012 / 0045436, US 2014 / 0178936, US 2017 / 0022282, or International Patent Publication WO 2006 / 113909, e.g., a humanized 1F6 variant.

[0141] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs or a light chain variable region comprising the three CDRs of the anti-CD70 antibody borsetuzumab. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs and a light chain variable region comprising the three CDRs of the anti-CD70 antibody borsetuzumab. In some embodiments, the CDRs are defined by the Kabat numbering scheme.

[0142] In some embodiments, the anti-CD70 antibody comprises the heavy chain variable region or the light chain variable region of the anti-CD70 antibody borsetuzumab. In some embodiments, the anti-CD70 antibody comprises the heavy chain variable region and the light chain variable region of the anti-CD70 antibody borsetuzumab.

[0143] In some embodiments, the anti-CD70 antibody is borsetuzumab.

[0144] Anti-CD70 antibodies of the invention may also be described or designated in terms of their binding affinity to CD70 (e.g., human CD70). Preferred binding affinities include those with a binding affinity of 5×10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 A dissociation constant or K less than M D These include those having the following structure:

[0145] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants, called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs vol. 1, no. 4, pp. 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0146] In some embodiments, the anti-CD70 antibody comprises a heavy chain variable domain as in any of the embodiments provided above and a light chain variable domain as in any of the embodiments provided above. a heavy chain constant region comprising the amino acid sequence of AS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK (SEQ ID NO: 5); TVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 6), including post-translational modifications of those sequences.

[0147] Antibodies also include derivatives that have been modified, i.e., by the covalent attachment of any type of molecule to the antibody, provided that the covalent attachment does not prevent the antibody from binding to CD70 or from exerting a cytostatic or cytotoxic effect on the cell. For example, without limitation, antibody derivatives include antibodies that have been modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, derivatives may contain one or more non-classical amino acids.

[0148] CD70-binding agents can optionally include an antibody effector domain that mediates or stimulates an ADCC, ADCP, and / or CDC response against CD70-expressing target cells. The effector domain can be, for example, the Fc domain(s) of an Ig molecule. Such CD70-binding agents can exert cytotoxic or cytostatic effects on CD70-expressing cancer cells, or can exert cytotoxic, cytostatic, or immunomodulatory effects on activated lymphocytes or dendritic cells, for example, in the treatment of CD70-expressing cancers or immunological disorders, respectively. Typically, CD70-binding agents recruit and / or activate cytotoxic leukocytes (e.g., natural killer (NK) cells, phagocytes (e.g., macrophages), and / or serum complement components).

[0149] The anti-CD70 antibody can be a humanized antibody, a single chain antibody, scFv, diabody, Fab, minibody, scFv-Fc, Fv, etc. In some embodiments, the CD70 antigen-binding region comprises an effector domain(s), e.g., the hinge-C of an immunoglobulin. H 2-C H Antigen-binding antibody fragments, including single-chain antibodies, can be prepared by combining, for example, the variable region with the whole or part of an effector domain (e.g., C H 2 and / or C H 3 domains alone or C H 1. Hinge and / or C L In some embodiments, the anti-CD70 antibody may comprise a hinge-C domain (in combination with a nucleotide sequence). The antigen-binding fragment may also comprise any combination of effector domains. H 2-C H It may be a single chain antibody comprising a CD70-binding variable region connected to three domains.

[0150] The effector domain of an anti-CD70 antibody can be derived from any suitable human immunoglobulin isotype. For example, the ability of human immunoglobulins to mediate CDC and ADCC / ADCP generally follows the order IgM ≈ IgG1 ≈ IgG3 > IgG2 > IgG4 and IgG1 ≈ IgG3 > IgG2 / IgM / IgG4, respectively. CD70-binding polypeptides can be expressed as recombinant fusion proteins containing appropriate constant domains that confer the desired effector functions. Upon binding to target cells, anti-CD70 antibodies or derivatives can trigger target cell destruction in vitro and in vivo via antibody effector functions, such as ADCC, CDC, and ADCP.

[0151] The CD70-binding agent can optionally be conjugated to a therapeutic agent, e.g., a cytotoxic, cytostatic, or immunomodulatory agent. Useful classes of cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like. In some exemplary embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycin, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholine-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone. In certain embodiments, the cytotoxic or cytostatic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof. Common auristatin E derivatives are, for example, esters formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other common auristatin derivatives include AFP, MMAF, and MMAE.The synthesis and structure of auristatin E and its derivatives are described in U.S. Patent Application Publication Nos. 20030083263 and 20050009751, International Patent Application No. PCT / US03 / 24209, International Patent Application No. PCT / US02 / 13435, and U.S. Patent Nos. 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, The cytotoxic agent is described in US Patent Nos. 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414. In certain embodiments, the cytotoxic agent is a DNA minor groove binder (see, for example, US Patent No. 6,130,237). For example, in some embodiments, the minor groove binder is a CBI compound. In other embodiments, the minor groove binder is an enediyne (e.g., calicheamicin). Examples of antitubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, and AEVB). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine, and colcemid, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatins, discodermolide, and ereutherobin. In some embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents.For example, in certain embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).

[0152] In some embodiments, the anti-CD70 antibody may be chimeric, comprising a human or non-human Fc region or portion thereof. For example, the antibody may comprise an Fc domain or portion of a non-human origin, such as a rodent (e.g., mouse or rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, chicken, or monkey (e.g., macaque, rhesus, etc.).

[0153] Anti-CD70 binding agents, e.g., antibodies, can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of CD70 and / or can be specific for both CD70 and for heterologous proteins (see, e.g., PCT Publications WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553). Multispecific antibodies, including bispecific and trispecific antibodies, useful for practicing the methods described herein are antibodies that immunospecifically bind to both CD70 (including, but not limited to, antibodies having the CDRs of monoclonal antibody 1F6) and a second cell surface receptor or receptor complex that mediates ADCC, ADCP, and / or CDC, e.g., CD16 / FcγRIII, CD64 / FcγRI, killer inhibitory or activating receptors, or the complement regulatory protein CD59. In some embodiments, binding of the multispecific antibody portion to the second cell surface molecule or receptor complex may enhance the effector function of the anti-CD70 antibody or other CD70-binding agent.

[0154] Antibodies can be produced by methods known in the art. For example, monoclonal antibodies can be prepared using a variety of techniques, including, for example, the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Hybridoma technology is generally discussed, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) and Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, NY, 1981).Examples of phage display methods that can be used to generate anti-CD70 antibodies include, for example, Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise of monoclonal antibodies as therapeutics in Anti-IgE and Allergic Disease, Jardieu and Fick Jr. (eds.), Marcel Dekker, New York, NY, Chapter 20, pp. 427-469; Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958, Persic et al., 1997, Gene 187:9-18, Burton et al., 1994, Advances in Immunology 57:191-280, PCT Application No. PCT / GB91 / 01134, PCT Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and those discussed in U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108, the disclosures of which are incorporated herein by reference.

[0155] Examples of techniques that can be used to produce single chain antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, Huston et al., 1991, Methods in Enzymology 203:46-88, Shu et al., 1993, Proc. Natl. Acad. Sci. USA 90:7995-7999, and Skerra et al., 1988, Science 240:1038-1040.

[0156] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with the two chains having different specificities (see, e.g., Milstein et al., 1983, Nature 305:537-39). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, some of which have the correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93 / 08829 and in Traunecker et al., 1991, EMBO J. 10:3655-59.

[0157] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion usually comprises a hinge, C H 2 and C H In some embodiments, the fusion is with an immunoglobulin heavy-chain constant domain comprising at least part of three regions. In some embodiments, the fusion has a first heavy-chain constant region (C) containing the site necessary for light-chain binding present in at least one of the fusions. H1). Nucleic acids having sequences encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, where expression of equal ratios of at least two polypeptide chains results in high yields or where the ratio is not particularly critical, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0158] In an embodiment of this approach, the bispecific antibody has a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of immunoglobulin light chains in only one-half of the bispecific molecules provides a facile separation method (see, e.g., International Publication No. WO 94 / 04690, incorporated herein by reference in its entirety).

[0159] For further discussion of bispecific antibodies, see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210; Rodrigues et al., 1993, J. Immunology 151:6954-61; Carter et al., 1992, Bio / Technology 10:163-67; Carter et al., 1995, J. Hematotherapy 4:463-70; Merchant et al., 1998, Nature Biotechnology 16:677-81. Using such techniques, bispecific antibodies can be prepared for use in the treatment or prevention of diseases as defined herein.

[0160] Bifunctional antibodies are also described in European Patent Publication No. EPA 0105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived biologically, i.e., by cell fusion techniques, or chemically, particularly using cross-linking or disulfide bridge-forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed, for example, in International Publication WO 83 / 03679 and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.

[0161] In some embodiments, framework residues in the human framework regions are substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by identifying framework residues important for antigen binding by modeling the interactions of CDR and framework residues and identifying unusual framework residues at specific positions by sequence comparison (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., 1988, Nature 332:323). Antibodies can be modified by, for example, CDR grafting (see, e.g., EP 0 239 400, PCT Publication WO 91 / 09967, U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (see, e.g., EP 0592 106, EP 0 519 596; Padlan, 1991, Molecular Immunology 28 (4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7 (6):805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973) and chain shuffling (see, e.g., U.S. Pat. No. 5,565,332) (all of these references are incorporated herein by reference).

[0162] Humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, as described in International Publication No. WO 87 / 02671, European Patent Publication No. 0 184 187, European Patent Publication No. 0 171 496, European Patent Publication No. 0 173 494, International Publication No. WO 86 / 01533, U.S. Pat. No. 4,816,567, European Patent Publication No. 0 012 023, Berter et al., 1988, Science 240:1041-43; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-43; Liu et al., 1987, J. Immunol. 139:3521-26; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-43; Liu et al., 1987, J. Immunol. 139:3521-26; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-43; USA 84:214-18; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-59; Morrison, 1985, Science 229:1202-07; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-25; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-60.

[0163] As noted above, a CD70-binding agent can be a derivative of an anti-CD70 antibody. Generally, an anti-CD70 antibody derivative comprises an anti-CD70 antibody (e.g., including an antigen-binding fragment or a conservatively substituted polypeptide) and at least one polypeptide region or other moiety heterologous to the anti-CD70 antibody. For example, an anti-CD70 antibody can be modified, e.g., by the covalent attachment of any type of molecule. Typical modifications include, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand (e.g., an albumin-binding molecule) or other protein, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0164] In some embodiments, the covalent bond does not interfere with effector function, e.g., it does not prevent the antibody derivative from specifically binding to CD70 via the antigen-binding region or a region derived therefrom, or the effector domain from specifically binding to an Fc receptor.

[0165] In some embodiments, the antibody derivative is a multimer, such as a dimer comprising one or more monomers, each monomer comprising (i) an antigen-binding region of an anti-CD70 antibody or a polypeptide region derived therefrom (e.g., by conservative substitution of one or more amino acids), and (ii) a multimerization (e.g., dimerization) polypeptide region such that the antibody derivative forms multimers (e.g., homodimers) that specifically bind to CD70. In typical embodiments, the antigen-binding region of an anti-CD70 antibody or a polypeptide region derived therefrom is recombinantly or chemically fused to a heterologous protein, and the heterologous protein comprises a dimerization or multimerization domain. Prior to administration of the antibody derivative to a subject for the treatment or prevention of an immunological disorder or CD70-expressing cancer, the derivative is subjected to conditions that allow for the formation of homodimers or heterodimers. A heterodimer, as used herein, can comprise the same dimerization domain but different CD70 antigen-binding regions, the same CD70 antigen-binding region but different dimerization domains, or different CD70 antigen-binding regions and dimerization domains.

[0166] Exemplary dimerization domains originate from transcription factors. In one embodiment, the dimerization domain is a basic region leucine zipper ("bZIP") (see Vinson et al., 1989, Science 246:911-916). Useful leucine zipper domains include, for example, those of the yeast transcription factor GCN4, the mammalian transcription factor CCAAT / enhancer-binding protein C / EBP, and the nuclear transformation in oncogene products Fos and Jun (see, e.g., Landschultz et al., 1988, Science 240:1759-64; Baxevanis and Vinson, 1993, Curr. Op. Gen. Devel. 3:278-285; O'Shea et al., 1989, Science 243:538-542). In another embodiment, the dimerization domain is that of a basic region helix-loop-helix ("bHLH") protein (see, e.g., Murre et al., 1989, Cell 56:777-783; see also Davis et al., 1990, Cell 60:733-746; Voronova and Baltimore, 1990, Proc. Natl. Acad. Sci. USA 87:4722-26). Particularly useful hHLH proteins include myc, max, and mac.

[0167] In still other embodiments, the dimerization domain is an immunoglobulin constant region, such as a heavy chain constant region or domain thereof (e.g., C H 1 domain, C H 2 domain and / or C H 3 domains) (see, e.g., U.S. Patent Nos. 5,155,027, 5,336,603, 5,359,046 and 5,349,053, EP 0 367 166 and WO 96 / 04388).

[0168] Heterodimers have been shown to form between Fos and Jun (Bohmann et al., 1987, Science 238:1386-1392), between members of the ATF / CREB family (Hai et al., 1989, Genes Dev. 3:2083-2090), between members of the C / EBP family (Cao et al., 1991, Genes Dev. 5:1538-52; Williams et al., 1991, Genes Dev. 5:1553-67; Roman et al., 1990, Genes Dev. 4:1404-15), and between ATF / CREB and members of the Fos / Jun family (Hai and Curran, 1991, Proc. Natl. Acad. Sci. USA 88:3720-24). Thus, when a CD70 binding protein is administered to a subject as a heterodimer comprising different dimerization domains, any combination of the foregoing may be used.

[0169] In other embodiments, the anti-CD70 antibody derivative is an anti-CD70 antibody conjugated to a second antibody (an "antibody heteroconjugate") (see, e.g., U.S. Pat. No. 4,676,980). Heteroconjugates useful for practicing the present methods include an antibody that binds to CD70 (e.g., an antibody having the CDRs and / or heavy chains of monoclonal antibody 1F6) and an antibody that binds to a surface receptor or receptor complex that mediates ADCC, phagocytosis, and / or CDC, e.g., CD16 / FcgRIII, CD64 / FcgRI, a killer cell activating or inhibitory receptor, or the complement control protein CD59. In exemplary embodiments, binding of the multispecific antibody portion to the second cell surface molecule or receptor complex enhances the effector function of the anti-CD70 antibody. In other embodiments, the antibody can be a therapeutic agent. Suitable antibody therapeutic agents are described herein.

[0170] In some embodiments, any of the anti-CD70 antibodies described herein are non-fucosylated.

[0171] In some embodiments, provided herein are populations of anti-CD70 antibodies comprising a plurality of anti-CD70 antibodies as described herein, wherein the anti-CD70 antibodies in the population of anti-CD70 antibodies have reduced core fucosylation. In some embodiments, at least 20% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 30% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 40% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 50% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 60% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 70% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 80% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 90% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 95% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 98% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 99% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, at least 99.5% of the antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, substantially none (i.e., less than 0.5%) of the antibodies in the population of anti-CD70 antibodies have core fucosylation. In some embodiments, all of the antibodies in the population of anti-CD70 antibodies lack core fucosylation.

[0172] Altered antibody glycosylation can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery, as described in U.S. Patent No. 10,196,445. Cells with altered glycosylation machinery have been described and can be used as host cells to express the recombinant antibodies of the disclosure, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α-(1,6) fucosyltransferase (see U.S. Patent Application Publication No. 20040110704, Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87:614); as a result, antibodies expressed in these cell lines lack fucose on their carbohydrates. As another example, EP 1176195 also describes cell lines with a functionally disrupted FUT8 gene, as well as cell lines with little or no activity to add fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO PCT Publication No. WO 2006 / 089231 describes a variant CHO cell line, Lec13, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates and results in hypofucosylation of antibodies expressed in the host cells. See also Shields et al. (2002) J. Biol. Chem. 277:26733. As described in PCT Publication No. WO 2006 / 089231, antibodies with modified glycosylation profiles can also be produced in chicken eggs. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, e.g., Lemna. See also, e.g., U.S. Publication No. 2012 / 0276086. PCT Publication No. WO 99 / 54342 describes cell lines that are engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies.See also Umana et al. (1999) Nat. Biotech. 17:176. Alternatively, fucose residues on antibodies may be cleaved off using a fucosidase enzyme. For example, the enzyme alpha-L-fucosidase removes fucosyl residues from antibodies. Tarentino et al. (1975) Biochem. 14:5516. Antibodies with reduced core fucosylation can be prepared by producing the antibody in a cell line that has been engineered to reduce core fucosylation using gene knockout, gene knockin, or RNAi. Small molecule inhibitors acting on enzymes in the glycosylation pathway can also be used to generate antibodies with reduced core fucosylation. Such methods are described in U.S. Patent No. 8,163,551. In some embodiments, an anti-CD70 antibody as described herein with reduced core fucosylation is produced by culturing a host cell expressing the antibody in a culture medium containing an effective amount of a fucose analog that reduces the incorporation of fucose into complex N-glycoside-linked sugar chains of the antibody or antibody derivative produced by the host cell. See U.S. Patent No. 8,163,551. Methods for producing nonfucosylated antibodies are also described in Pereira et al. (2018) MAbs 10(5):693-711.

[0173] In some embodiments, the anti-CD70 antibody or derivative thereof competitively inhibits the binding of mAb 1F6 to CD70, as determined by any method known in the art for determining competitive binding (e.g., an immunoassay described herein). In typical embodiments, the antibody competitively inhibits the binding of 1F6 to CD70 by at least 50%, at least 60%, at least 70%, or at least 75%. In other embodiments, the antibody competitively inhibits the binding of 1F6 to CD70 by at least 80%, at least 85%, at least 90%, or at least 95%.

[0174] Antibodies can be assayed for specific binding to CD70 by any of a variety of known methods. Immunoassays that can be used include competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometry, fluorescence immunoassay, and protein A immunoassay. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds., Short Protocols in Molecular Biology (John Wiley and Sons, Inc., New York, 4th ed. 1999); Harlow and Lane, Using Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999)).

[0175] Furthermore, the binding affinity of an antibody to CD70 and the off-rate of the antibody-CD70 interaction can be determined by competitive binding assays. As an example of a competitive binding assay, labeled CD70 (e.g., 3 H or 125 A radioimmunoassay involves incubating I) with an antibody of interest in the presence of increasing amounts of unlabeled CD70 and detecting antibody bound to the labeled CD70. The affinity of the antibody for CD70 and the binding off-rate can then be determined from the data by Scatchard plot analysis. Competition with a second antibody (e.g., mAb 1F6) can also be determined using a radioimmunoassay. In this case, CD70 binds to a labeled compound (e.g., 3 H or 125The antibody of interest conjugated to I) is incubated in the presence of increasing amounts of an unlabeled second antibody. Alternatively, the binding affinity of the antibody to CD70 and the on-rate and off-rate of the antibody-CD70 interaction can be determined by surface plasmon resonance. In some embodiments, the anti-CD70 antibody or derivative thereof can be targeted and accumulated at the membrane of CD70-expressing cells.

[0176] Anti-CD70 antibodies and derivatives thereof can be produced by methods known in the art for the synthesis of proteins, typically by, for example, recombinant expression techniques. Recombinant expression of an antibody or derivative thereof that binds to CD70 typically involves construction of an expression vector containing a nucleic acid encoding the antibody or derivative thereof. Vectors for the production of protein molecules can be produced by recombinant DNA technology using techniques known in the art. Standard techniques, such as those described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001), Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2nd ed., 1989), Short Protocols in Molecular Biology (Ausubel et al., John Wiley and Sons, New York, 4th ed., 1999), and Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, DC, 2nd ed., 1998), can be used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture, transgene integration, and recombinant protein expression.

[0177] For example, for recombinant expression of an anti-CD70 antibody, an expression vector can encode the heavy or light chain or heavy or light chain variable domain thereof operably linked to a promoter. Expression vectors can include, for example, nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., PCT Publication WO 86 / 05807, PCT Publication WO 89 / 01036, and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain. The expression vector is transferred into host cells by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the anti-CD70 antibody. In a typical embodiment for expression of a double-chain antibody, vectors encoding both the heavy and light chains can be co-expressed in host cells for expression of the entire immunoglobulin molecule.

[0178] A variety of prokaryotic and eukaryotic host-expression vector systems can be utilized to express anti-CD70 antibodies or derivatives thereof. Typically, eukaryotic cells are used for the expression of recombinant proteins, particularly for recombinant whole anti-CD70 antibody molecules. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for the production of anti-CD70 antibodies and derivatives thereof (see, e.g., Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio / Technology 8:2).

[0179] Other host-expression systems include, for example, plasmid-based expression systems in bacterial cells (see, e.g., Ruther et al., 1983, EMBO 1,2:1791; Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509), insect systems such as the use of Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors in Spodoptera frugiperda cells, and viral-based expression systems in mammalian cells, such as adenovirus-based systems (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359; Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0180] Additionally, a host cell line can be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing (e.g., glycosylation, phosphorylation, and cleavage) of the expressed protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript and gene product can be used. Such mammalian host cells include, for example, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.

[0181] For long-term, high-yield production of recombinant anti-CD70 antibodies or derivatives thereof or other CD70-binding agents, stable expression systems are typically used. For example, cell lines stably expressing anti-CD70 antibodies or derivatives thereof can be engineered by transforming host cells with DNA controlled by appropriate expression control elements (e.g., promoter and enhancer sequences, transcription terminators, polyadenylation sites) and a selectable marker, followed by growth of the transformed cells in a selective medium. The selectable marker confers resistance to selection and allows cells to stably integrate the DNA into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. For example, tk and tk+, respectively, can be used to express the anti-CD70 antibodies or derivatives thereof. - , hgprt - or aprt - Several selection systems are available that can be used in cells, including the herpes simplex virus thymidine kinase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes. Additionally, the following genes can be used as the basis for selection: antimetabolite resistance, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin. Methods generally known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley and Sons, NY, 1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual (Stockton Press, NY, 1990), Current Protocols in Human Genetics (Dracopoli et al., eds., John Wiley and Sons, NY, 1994, Chapters 12 and 13), and Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.

[0182] The expression level of the antibody or derivative can be increased by vector amplification (see generally, e.g., Bebbington and Hentschel, The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the anti-CD70 antibody or derivative thereof is amplifiable, host cells with increased copy numbers of the marker gene conferring resistance to the inhibitor are selected by increasing the level of the inhibitor present in the host cell culture medium. The copy number of the associated antibody gene also increases, thereby increasing expression of the antibody or derivative thereof (see Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0183] When an anti-CD70 antibody contains both a heavy chain and a light chain or derivatives thereof, the host cell may be co-transfected with two expression vectors: one encoding the heavy chain protein and the second encoding the light chain protein. The two vectors may contain identical selectable markers that allow equal expression of the heavy and light chain proteins. Alternatively, a single vector may be used that encodes and is capable of expressing both the heavy and light chain proteins. In such a situation, the light chain is usually placed before the heavy chain to avoid excess non-toxic heavy chain (see Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0184] Once an anti-CD70 antibody or derivative thereof is produced (e.g., by animal, chemical synthesis, or recombinant expression), it can be purified by any suitable method for protein purification, including, for example, chromatography (e.g., ion exchange or affinity chromatography (e.g., protein A chromatography for purification of antibodies with an intact Fc region)), centrifugation, differential solubility, or any other standard technique for protein purification. The anti-CD70 antibody or derivative thereof can be fused to, for example, a marker sequence, e.g., a peptide, to facilitate purification by affinity chromatography. Suitable marker amino acid sequences include, for example, a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, CA 91311), and the "HA" tag (Wilson et al., 1984, Cell 37:767) and "flag" tag, which correspond to an epitope derived from the influenza hemagglutinin protein.

[0185] Once an anti-CD70 antibody or derivative thereof is generated, its ability to exert a cytostatic or cytotoxic effect on CD70-expressing cancer cells or an immunomodulatory effect on CD70-expressing immune cells is determined by methods described below or known in the art.

[0186] To minimize the activity of anti-CD70 antibodies on the outside of activated immune cells or CD70-expressing cancer cells, antibodies that specifically bind to cell membrane-bound CD70 but do not specifically bind to soluble CD70 can be used to concentrate the anti-CD70 antibodies on the cell surface of activated immune cells or CD70-expressing cancer cells.

[0187] Typically, an anti-CD70 antibody or derivative is substantially purified (e.g., substantially free from substances that limit its effectiveness or that produce undesirable side effects). In some embodiments, the anti-CD70 antibody or derivative is at least about 40% pure, at least about 50% pure, or at least about 60% pure. In some embodiments, the anti-CD70 antibody or derivative is at least about 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure. In some embodiments, the anti-CD70 antibody or derivative is approximately 99% pure.

[0188] III. Treatment Methods The present invention provides a method of treating a CD70-expressing cancer, e.g., a myeloid malignancy, in a subject, comprising administering to the subject a therapeutically effective amount of an anti-CD70 antibody, e.g., a nonfucosylated anti-CD70 antibody, described herein. Myeloid malignancies include acute myeloid leukemia (AML), myeloproliferative disorders (MPDS), myelodysplastic syndromes (MDS), and myelodysplastic / myeloproliferative syndromes, all of which are clonal stem cell (HSC) or precursor malignant disorders. In some embodiments, the cancer is MDS. In some embodiments, the cancer is AML. MDS encompasses multiple subtypes, including MDS with single-lineage dysplasia, MDS with ringed sideroblasts, MDS with multilineage dysplasia, MDS with excess blasts, MDS with isolated del(5q), and MDS, unclassified. MDS is characterized by ineffective hematopoiesis in one or more myeloid lineages. Early-stage MDS often demonstrates excessive apoptosis and hematopoietic cell dysplasia. In approximately one-third of MDS patients, this ineffective hematopoiesis precedes progression to secondary AML (sAML). AML is a malignant tumor of the myeloid lineage of white blood cells. In some embodiments, a method comprises administering to a subject a therapeutically effective amount of a nonfucosylated anti-CD70 antibody, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, where the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme. In some embodiments, a method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 30% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, a method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 40% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to the subject a population of anti-CD70 antibodies, wherein at least 50% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation, hi some embodiments, the method comprises administering to the subject a population of anti-CD70 antibodies, wherein at least 60% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation.In some embodiments, the method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 70% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 80% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 90% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 95% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to a subject a population of anti-CD70 antibodies, wherein at least 98% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to the subject a population of anti-CD70 antibodies, wherein at least 99% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the method comprises administering to the subject a population of anti-CD70 antibodies, wherein at least 99.5% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. In some embodiments, the anti-CD70 antibody is administered in combination with a hypomethylating agent (HMA). In some embodiments, the HMA is azacytidine. In some embodiments, the anti-CD70 antibody is administered in combination with a BH3 mimetic. In some embodiments, the anti-CD70 antibody is administered in combination with venetoclax (VENCLEXTA®). In some embodiments, the anti-CD70 antibody is administered in combination with an HMA and a BH3 mimetic. In some embodiments, the anti-CD70 antibody is administered in combination with an HMA and venetoclax. In some embodiments, the anti-CD70 antibody is administered in combination with azacitidine and a BH3 mimetic. In some embodiments, the anti-CD70 antibody is administered in combination with azacitidine and venetoclax.

[0189] In some embodiments, provided herein are methods of treating CD70-expressing MDS in a subject, the methods comprising administering a therapeutically effective amount of an anti-CD70 antibody described herein. In some embodiments, the anti-CD70 antibody is non-fucosylated. In some embodiments, the MDS is relapsed or refractory MDS. In some embodiments, the MDS is relapsed MDS. In some embodiments, the MDS is refractory MDS. In some embodiments, the subject has experienced treatment failure after previous hypomethylating agent (HMA) therapy for MDS. HMAs (also known as dimethylating agents) are drugs that inhibit DNA methylation. In some embodiments, the HMA is a DNA methyltransferase inhibitor. In some embodiments, the HMA is azacitidine. In some embodiments, the HMA is decitabine.

[0190] In some embodiments, provided herein are methods of treating CD70-expressing AML in a subject, the methods comprising administering a therapeutically effective amount of an anti-CD70 antibody described herein. In some embodiments, the anti-CD70 antibody is non-fucosylated. In some embodiments, the AML is relapsed or refractory AML. In some embodiments, the AML is relapsed AML. In some embodiments, the AML is refractory AML. In some embodiments, the subject has received one previous treatment regimen to treat the AML. In some embodiments, the subject has received two previous treatment regimens to treat the AML. In some embodiments, the subject has received three previous treatment regimens to treat the AML.

[0191] In some embodiments, at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of cancer cells from a subject express CD70. In some embodiments, at least 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, or at least 80% of cancer cells from a subject express CD70. In some embodiments, the percentage of cells expressing CD70 is determined using immunohistochemistry (IHC). In some embodiments, the percentage of cells expressing CD70 is determined using flow cytometry. In some embodiments, the percentage of cells expressing CD70 is determined using enzyme-linked immunosorbent assay (ELISA).

[0192] In one aspect, a method of treating cancer with an anti-CD70 antibody as described herein results in an improvement in one or more therapeutic effects in a subject following administration of the antibody relative to baseline. In some embodiments, the one or more therapeutic effects are objective response rate, duration of response, time to response, progression-free survival, overall survival, or any combination thereof. In one embodiment, the one or more therapeutic effects are stable disease. In one embodiment, the one or more therapeutic effects are partial responses. In one embodiment, the one or more therapeutic effects are complete responses. In one embodiment, the one or more therapeutic effects are objective response rates. In one embodiment, the one or more therapeutic effects are duration of response. In one embodiment, the one or more therapeutic effects are time to response. In one embodiment, the one or more therapeutic effects are progression-free survival. In one embodiment, the one or more therapeutic effects are overall survival. In one embodiment, the one or more therapeutic effects are cancer regression.

[0193] In one embodiment of the methods or uses or products for use provided herein, the response to treatment with an anti-CD70 antibody as described herein may include the following criteria (Cheson criteria):

[0194] TIFF0007824220000001.tif236169TIFF0007824220000002.tif15168

[0195] In one embodiment of the methods or uses or products for use provided herein, the response to treatment with an anti-CD70 antibody as described herein may include the following criteria (Cheson criteria):

[0196] TIFF0007824220000003.tif235169TIFF0007824220000004.tif53168

[0197] TIFF0007824220000005.tif109169

[0198] In one embodiment of the methods or uses or products for use provided herein, the efficacy of treatment with an anti-CD70 antibody as described herein is assessed by measuring the objective response rate. In some embodiments, the objective response rate is the percentage of patients experiencing a reduction in tumor size by a predefined amount and for a minimum duration. In some embodiments, the objective response rate is based on the Cheson criteria. In one embodiment, the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In one embodiment, the objective response rate is at least about 20% to 80%. In one embodiment, the objective response rate is at least about 30% to 80%. In one embodiment, the objective response rate is at least about 40% to 80%. In one embodiment, the objective response rate is at least about 50% to 80%. In one embodiment, the objective response rate is at least about 60% to 80%. In one embodiment, the objective response rate is at least about 70% to 80%. In one embodiment, the objective response rate is at least about 80%. In one embodiment, the objective response rate is at least about 85%. In one embodiment, the objective response rate is at least about 90%. In one embodiment, the objective response rate is at least about 95%. In one embodiment, the objective response rate is at least about 98%. In one embodiment, the objective response rate is at least about 99%. In one embodiment, the objective response rate is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, or at least 80%. In one embodiment, the objective response rate is at least 20% to 80%. In one embodiment, the objective response rate is at least 30% to 80%. In one embodiment, the objective response rate is at least 40% to 80%. In one embodiment, the objective response rate is at least 50% to 80%. In one embodiment, the objective response rate is at least 60% to 80%. In one embodiment, the objective response rate is at least 70% to 80%. In one embodiment, the objective response rate is at least 80%.In one embodiment, the objective response rate is at least 85%. In one embodiment, the objective response rate is at least 90%. In one embodiment, the objective response rate is at least 95%. In one embodiment, the objective response rate is at least 98%. In one embodiment, the objective response rate is at least 99%. In one embodiment, the objective response rate is 100%.

[0199] In one embodiment of the methods or uses or products for use described herein, response to treatment with an anti-CD70 antibody as described herein is assessed by measuring the time to progression-free survival after administration of an anti-CD70 antibody as described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of an anti-CD70 antibody as described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 6 months after administration of an anti-CD70 antibody as described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 1 year after administration of an anti-CD70 antibody as described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 2 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 3 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 4 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least about 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least 6 months after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits at least one year of progression-free survival after administration of an anti-CD70 antibody described herein.In some embodiments, the subject exhibits a progression-free survival of at least two years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least three years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least four years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits a progression-free survival of at least five years after administration of an anti-CD70 antibody described herein.

[0200] In one embodiment of the methods or uses or products for use described herein, response to treatment with an anti-CD70 antibody described herein is assessed by measuring the time to overall survival after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 6 months after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 1 year after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 2 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 3 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 4 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least about 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least about 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least 6 months after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least 1 year after administration of an anti-CD70 antibody described herein.In some embodiments, the subject exhibits an overall survival of at least two years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least three years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least four years after administration of an anti-CD70 antibody described herein. In some embodiments, the subject exhibits an overall survival of at least five years after administration of an anti-CD70 antibody described herein.

[0201] In one embodiment of the methods or uses or products for use described herein, the response to treatment with an anti-CD70 antibody described herein is assessed by measuring the duration of response to the anti-CD70 antibody described herein after administration of the anti-CD70 antibody described herein. In some embodiments, the duration of response to the anti-CD70 antibody described herein is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the anti-CD70 antibody described herein. In some embodiments, the duration of response to the anti-CD70 antibody described herein is at least about 6 months after administration of the anti-CD70 antibody described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least about 1 year after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least about 2 years after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least about 3 years after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least about 4 years after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least about 5 years after administration of the anti-CD70 antibodies described herein.In some embodiments, the duration of response to an anti-CD70 antibody described herein is at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of an anti-CD70 antibody described herein. In some embodiments, the duration of response to an anti-CD70 antibody described herein is at least 6 months after administration of an anti-CD70 antibody described herein. In some embodiments, the duration of response to an anti-CD70 antibody described herein is at least 1 year after administration of an anti-CD70 antibody described herein. In some embodiments, the duration of response to an anti-CD70 antibody described herein is at least 2 years after administration of an anti-CD70 antibody described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least 3 years after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least 4 years after administration of the anti-CD70 antibodies described herein. In some embodiments, the duration of response to the anti-CD70 antibodies described herein is at least 5 years after administration of the anti-CD70 antibodies described herein.

[0202] In some embodiments of the methods or uses or products for use described herein, administering to a subject an anti-CD70 antibody described herein, e.g., a nonfucosylated anti-CD70 antibody, results in depletion of cancer cells in the subject. In some embodiments, administering an anti-CD70 antibody described herein, e.g., a nonfucosylated anti-CD70 antibody, results in depletion of cancer cells by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 5% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 10% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 20% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 30% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 40% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 50% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 60% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 70% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 80% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least about 90% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject.In some embodiments, cancer cells are depleted by at least about 95% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, cancer cells are depleted by at least about 99% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, cancer cells are depleted by about 100% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, administering an anti-CD70 antibody described herein, e.g., a nonfucosylated anti-CD70 antibody, results in at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least about 80%, at least about 90%, at least 95%, or 100% depletion of cancer cells compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 5% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 10% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 20% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 30% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 40% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 50% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 60% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 70% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are depleted by at least 80% compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject.In some embodiments, the cancer cells are at least 90% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 95% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are at least 99% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject. In some embodiments, the cancer cells are 100% depleted compared to the amount of cancer cells before administering the anti-CD70 antibody to the subject.

[0203] In some embodiments of the methods or uses or products for use described herein, administering to a subject an anti-CD70 antibody described herein, e.g., a nonfucosylated anti-CD70 antibody, does not result in depletion of CD70+ regulatory T cells (CD70+ Tregs) in the subject. In some embodiments, administering an anti-CD70 antibody described herein, e.g., a nonfucosylated anti-CD70 antibody, results in depletion of CD70+ Tregs by about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.1% or less compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by about 50% or less compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 40% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 30% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 20% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 10% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 5% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than about 1% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by about 0.1% or less compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, administering an anti-CD70 antibody described herein, e.g., a non-fucosylated anti-CD70 antibody, results in depletion of CD70+ Tregs by 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% or less compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject.In some embodiments, CD70+ Tregs are depleted by no more than 50% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 40% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 30% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 20% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 10% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 5% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 1% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject. In some embodiments, CD70+ Tregs are depleted by no more than 0.1% compared to the amount of CD70+ Tregs before administering the anti-CD70 antibody to the subject.

[0204] In some embodiments, a fucosylated anti-CD70 antibody depletes CD70+ Tregs in a subject to a greater extent than a nonfucosylated form of an anti-CD70 antibody comprising the same heavy and light chain amino acid sequences. In some embodiments, a fucosylated anti-CD70 antibody depletes CD70+ Tregs in a subject to a greater extent than a nonfucosylated form of an anti-CD70 antibody comprising the same heavy and light chain amino acid sequences when the subject is homozygous for the high-affinity FcγRIIIa receptor (V / V 158). In some embodiments, a fucosylated anti-CD70 antibody depletes CD70+ Tregs in a subject to the same extent as a nonfucosylated form of an anti-CD70 antibody comprising the same heavy and light chain amino acid sequences when the subject is homozygous for the low-affinity FcγRIIIa receptor (F / F 158). In some embodiments, when a subject is homozygous for the high affinity FcγRIIIa receptor (V / V 158), neither a fucosylated anti-CD70 antibody nor a nonfucosylated form of an anti-CD70 antibody comprising the same heavy and light chain amino acid sequences depletes CD8 T cells. In some embodiments, when a subject is homozygous for the low affinity FcγRIIIa receptor (F / F 158), neither a fucosylated anti-CD70 antibody nor a nonfucosylated form of an anti-CD70 antibody comprising the same heavy and light chain amino acid sequences depletes CD8 T cells.

[0205] IV. Assays for Cytotoxic, Cytostatic, and Immunomodulatory Activities Methods for determining whether an antibody mediates an effector function on a target cell are known, and illustrative examples of such methods are described below.

[0206] To determine whether an anti-CD70 antibody mediates antibody-dependent cellular cytotoxicity against activated immune cells or CD70-expressing cancer cells, an assay can be used that mediates target cell death in the presence of the antibody and effector immune cells. The assay used to measure this type of cytotoxicity involves the production of cytotoxicity from metabolically labeled target cells after incubation in the presence of effector cells and target-specific antibody. 51The method may be based on the determination of Cr release (see, e.g., Perussia and Loza, 2000, Methods in Molecular Biology 121:179-92 and "Cell Signaling in Immunology," in Current Protocols in Immunology, Coligan et al., eds., Wiley & Sons, 1993). 51 (See "Cr Release Assay of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)"). For example, Na2 51 Activated immune cells (e.g., activated lymphocytes) or CD70-expressing cancer cells labeled with CrO4 and plated at a density of 5,000 cells per well of a 96-well plate can be treated with varying concentrations of anti-CD70 antibodies for 30 minutes and then mixed with normal human peripheral blood mononuclear cells (PBMCs) for 4 hours. Membrane disruption accompanying target cell death results in 51 Cr is released into the culture supernatant, which can be collected and evaluated for radioactivity as a measure of cytotoxic activity. Other assays for measuring ADCC may involve non-radioactive labels or may be based on the induced release of specific enzymes. For example, a non-radioactive assay based on time-resolved fluorometry is commercially available (Delphia, Perkin Elmer). This assay is based on loading target cells with the acetoxymethyl ester of a fluorescence-enhancing ligand (BATDA), which permeates the cell membrane and then hydrolyzes to form a membrane-impermeable hydrophilic ligand (TDA). TDA is released from lysed cells when mixed with target-specific antibodies and PBMC effector cells and is available to form a highly fluorescent chelate when mixed with europium. The signal measured with a time-resolved fluorimeter correlates with the amount of cell lysis.

[0207] To determine whether an anti-CD70 antibody mediates antibody-dependent cellular phagocytosis of activated immune cells or CD70-expressing cancer cells, an assay measuring target cell internalization by effector immune cells (e.g., freshly cultured macrophages or established macrophage-like cell lines) can be used (see, e.g., Munn and Cheung, 1990, J. Exp. Med. 172:231-37; Keler et al., 2000, J. Immunol. 164:5746-52; Akewanlop et al., 2001, Cancer Res. 61:4061-65). For example, target cells can be labeled with a lipophilic membrane dye, e.g., PKH67 (Sigma), coated with target-specific antibody, and mixed with effector immune cells for 4 to 24 hours. Effector cells may then be identified by counterstaining with a fluorochrome-conjugated antibody specific for a phagocyte surface marker (e.g., CD14) and analyzing the cells by two-color flow cytometry or fluorescence microscopy. Double-positive cells represent effector cells that have been internalized by target cells. For these assays, effector cells may be monocytes derived from PBMCs that have been differentiated into macrophages by culture with M-CSF or GM-CSF for 5-10 days (see, e.g., Munn and Cheung, supra). The human macrophage-like cell lines U937 (Larrick et al., 1980, J. Immunology 125:6-12) or THP-1 (Tsuchiya et al., 1980, Int. J. Cancer 26:171-76), available from the American College of Cardiovascular Sciences (ATCC), may also be used as alternative sources of phagocytes.

[0208] Methods for determining whether an antibody mediates complement-dependent cytotoxicity upon binding to a target cell are also known. The same methods can be applied to determine whether an anti-CD70 antibody mediates CDC against activated immune cells or CD70-expressing cancer cells. Illustrative examples of such methods are described below.

[0209] The source of active complement can be normal human serum or purified from laboratory animals, including rabbits. In a standard assay, an anti-CD70 antibody is incubated with CD70-expressing activated immune cells (e.g., activated lymphocytes) or CD70-expressing cancer cells in the presence of complement. The ability of such anti-CD70 antibodies to mediate cell lysis can be determined by several readouts. In one example, Na 51 A CrO4 release assay is used. In this assay, target cells release Na 51 Labeled with CrO4. Unincorporated Na 51 The CrO4 is washed away, and the cells are plated at an appropriate density, typically between 5,000 and 50,000 cells / well in a 96-well plate. Incubation with anti-CD70 antibody in the presence of normal serum or purified complement typically lasts 2 to 6 hours at 37°C in a 5% CO2 atmosphere. The released radioactivity, indicating cell lysis, in an aliquot of the culture supernatant is determined by gamma counting. Maximum cell lysis is determined by the amount of Na incorporated by detergent (0.5 to 1% NP-40 or Triton X-100) treatment. 51The second readout is the reduction of a metabolic dye, e.g., Alamar Blue, by viable cells. In this assay, target cells are incubated with anti-CD70 antibody and complement and incubated as described above. At the end of the incubation, 1 / 10 volume of Alamar Blue (Biosource International, Camarillo, CA) is added. Incubation is continued for up to 16 hours at 37°C in a 5% CO2 atmosphere. The reduction of Alamar Blue, as an indicator of metabolically active viable cells, is determined by fluorometric analysis using excitation at 530 nm and emission at 590 nm. The third readout is cell membrane permeability to propidium iodide (PI). The formation of pores in the cell membrane as a result of complement activation facilitates the entry of PI into the cell, where it diffuses into the nucleus and binds to DNA. Upon binding to DNA, PI fluorescence at 600 nm increases significantly. Treatment of target cells with anti-CD70 antibody and complement is carried out as described above. At the end of the incubation, PI is added to a final concentration of 5 μg / ml. The cell suspension is then examined by flow cytometry using a 488 nm argon laser for excitation. Lysed cells are detected by fluorescence emission at 600 nm.

[0210] V. Pharmaceutical Compositions Comprising Anti-CD70 Antibodies and Their Administration Compositions comprising anti-CD70 antibodies can be administered to subjects having or at risk of having a CD70-expressing cancer. The present invention further provides for the use of anti-CD70 antibodies in the manufacture of a medicament for preventing or treating a CD70-expressing cancer. The term "subject," as used herein, means any mammalian patient to which a CD70-binding agent can be administered, including, for example, humans and non-human mammals, such as primates, rodents, and dogs. Subjects specifically intended for treatment using the methods described herein include humans. The antibodies can be administered alone or in combination with other compositions in the prevention or treatment of a CD70-expressing cancer.

[0211] Various delivery systems are known and can be used to administer anti-CD70 antibodies. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Anti-CD70 antibodies can be administered, for example, by infusion or bolus injection (e.g., intravenous or subcutaneous), by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents, such as chemotherapeutic agents. Administration can be systemic or local. In one embodiment, the anti-CD70 antibodies described herein are administered parenterally. Parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes epithelial, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion. In some embodiments, the route of administration of the anti-CD70 antibodies described herein is intravenous injection or infusion. In some embodiments, the route of administration of the anti-CD70 antibodies described herein is intravenous infusion.

[0212] In certain embodiments, the anti-CD70 antibody composition is administered by injection, catheter, suppository, or implant, which is a porous, non-porous, or gelatinous material, including, for example, a membrane, such as a sialastic membrane, or a fiber. Typically, the composition is administered using a material to which the anti-CD70 antibody does not absorb.

[0213] Anti-CD70 antibodies can be administered as pharmaceutical compositions containing a therapeutically effective amount of the antibody and one or more pharmaceutically compatible ingredients. For example, pharmaceutical compositions typically contain one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions may be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will typically contain a therapeutically effective amount of the protein in purified form, together with an amount of carrier appropriate to provide the form for proper administration to the patient. The formulation will depend on the mode of administration.

[0214] In a typical embodiment, the pharmaceutical composition is formulated according to conventional methods as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the pharmaceutical is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0215] Additionally, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing the anti-CD70 antibody in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent may be used for reconstitution or dilution of the lyophilized anti-CD70 antibody. Optionally, such container may be associated with a notice in a format prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice reflecting approval by the agency of manufacture, use, or sale for human administration.

[0216] The amount of anti-CD70 antibody that is effective in treating or preventing CD70-expressing cancer can be determined by standard clinical techniques. Additionally, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also vary depending on the route of administration and the stage of the CD70-expressing cancer, and must be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0217] For example, the toxicity and therapeutic efficacy of anti-CD70 antibodies are 50(the dose that is lethal to 50% of the population) and ED 50 The LD (the dose which is therapeutically effective in 50% of the population) can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Anti-CD70 antibodies that exhibit large therapeutic indices are preferred. In cases where the anti-CD70 antibody exhibits toxic side effects, a delivery system can be used that targets the anti-CD70 antibody to the site of affected tissue to minimize potential damage to non-CD70-expressing cells, thereby reducing side effects.

[0218] The data obtained from cell culture assays and animal studies can be used in formulating various dosages for use in humans. The dose of anti-CD70 antibodies is usually determined to be ED50 with little or no toxicity. 50 The therapeutically effective dose of an anti-CD70 antibody used in the present method can be estimated initially from cell culture assays. The dose can be determined based on the IC50 concentration, as determined in cell culture. 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0219] Typically, the dosage of an anti-CD70 antibody administered to a patient with a CD70-expressing cancer is about 0.1 mg / kg to 100 mg / kg of the subject's body weight. More typically, the dosage administered to a subject is 0.1 mg / kg to 50 mg / kg of the subject's body weight, and even more typically, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, 1 mg / kg to 10 mg / kg, or 1 mg / kg to 7.5 mg / kg of the subject's body weight. In some embodiments, the dose of the anti-CD70 antibody is 1.5 mg / kg. In some embodiments, the dose is 5 mg / kg. In some embodiments, the dose is 10 mg / kg. In some embodiments, the dose is 20 mg / kg. Generally, human antibodies have a longer half-life within the human body than antibodies derived from other species due to the immune response to foreign proteins. Therefore, lower and less frequent doses of anti-CD70 antibodies, including humanized or chimeric antibodies, are often possible.

[0220] Doses of anti-CD70 antibody can be administered, for example, daily, once a week (weekly), twice a week, three times a week, four times a week, five times a week, every other week, monthly, or otherwise as needed.

[0221] In some embodiments, the dose of the anti-CD70 antibody is a suboptimal dose (i.e., the EC 50 For example, the dosage of the anti-CD70 antibody can include a dosage selected from at least 25%, at least 15%, at least 10%, or at least 5% of the therapeutic window. As used herein, the term "therapeutic window" refers to the range of drug dosages or concentrations in a body system that provide a safe and effective therapy.

[0222] In some embodiments, the dose of anti-CD70 antibody is about 0.05 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 0.9 mg / kg, or about 0.15 to about 0.75 mg / kg of the subject's body weight. Such doses can be administered 1 to about 15 times per week. Each dose can be the same or different. For example, a dose of about 0.15 mg / kg of anti-CD70 antibody can be administered 1 to 10 times per 4, 5, 6, or 7 days.

[0223] In some embodiments, a pharmaceutical composition comprising an anti-CD70 antibody may further comprise a therapeutic agent (e.g., an unconjugated cytotoxic agent or immunomodulatory agent, such as any of those described herein). Anti-CD70 binding agents may also be co-administered in combination with one or more therapeutic agents for the treatment or prevention of CD70-expressing cancer. For example, the combination therapy may include a therapeutic agent (e.g., a cytostatic agent, a cytotoxic agent, or an immunomodulatory agent, e.g., an unconjugated cytostatic agent, a cytotoxic agent, or an immunomodulatory agent, e.g., one conventionally used for the treatment of cancer). The combination therapy may also include the administration of an agent that targets a receptor or receptor complex other than CD70 on the surface of, for example, activated lymphocytes, dendritic cells, or CD70-expressing cancer cells. Examples of such agents include a second, non-CD70 antibody that binds to a molecule on the surface of activated lymphocytes, dendritic cells, or CD70-expressing cancer cells. Another example includes a ligand that targets such a receptor or receptor complex. Typically, such antibodies or ligands bind to cell surface receptors on activated lymphocytes, dendritic cells, or CD70-expressing cancer cells and deliver a cytostatic or cytotoxic signal to the activated lymphocytes, dendritic cells, or CD70-expressing cancer cells, thereby enhancing the cytotoxic or cytostatic effect of the anti-CD70 antibody. Such combinatorial administration may have additive or synergistic effects on disease parameters (e.g., symptom severity, number of symptoms, or frequency of relapses). Another example includes hypomethylating agents (HMAs). In some embodiments, the HMA is azacytidine (VIDAZA®). Another example includes BH3 mimetics. Another example includes venetoclax (VENCLEXTA®). In some embodiments, a pharmaceutical composition comprises an anti-CD70 antibody, an HMA, and a BH3 mimetic. In some embodiments, a pharmaceutical composition comprises an anti-CD70 antibody, an HMA, and venetoclax. In some embodiments, the pharmaceutical composition comprises an anti-CD70 antibody, azacitidine, and a BH3 mimetic. In some embodiments, the pharmaceutical composition comprises an anti-CD70 antibody, azacitidine, and venetoclax.

[0224] With respect to treatment regimens for combinatorial administration, in certain embodiments, the anti-CD70 antibody is administered simultaneously with the therapeutic agent. In another specific embodiment, the therapeutic agent is administered at least 1 hour and up to several months before or after administration of the anti-CD70 antibody, e.g., at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, or 3 months before or after administration of the anti-CD70 antibody. In some embodiments, the subject is monitored after administration of the anti-CD70 antibody and optionally the therapeutic agent.

[0225] VI. Manufactured Articles and Kits In another aspect, an article of manufacture or kit is provided that includes an anti-CD70 antibody described herein. The article of manufacture or kit may further include instructions for using the anti-CD70 antibody described herein in the methods of the invention. Thus, in certain embodiments, the article of manufacture or kit includes instructions for using the anti-CD70 antibody described herein in a method of treating cancer (e.g., a myeloid malignancy) in a subject, comprising administering to the subject an effective amount of an anti-CD70 antibody described herein. In some embodiments, the cancer is MDS. In some embodiments, the cancer is AML. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the subject is a human.

[0226] The article of manufacture or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., double-chamber vials), syringes (e.g., single- or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container can be formed from a variety of materials, for example, glass or plastic. The container holds the formulation.

[0227] The article of manufacture or kit may further include a label or package insert, which may be on or associated with the container and include instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful for or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other mode of administration for treating cancer in a subject. The container holding the formulation may be a single-use vial or a multi-use vial that allows for repeated administration of the reconstituted formulation. The article of manufacture or kit may further include a second container containing a suitable diluent. The article of manufacture or kit may further include other materials desirable from commercial, therapeutic, and user standpoints, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0228] The articles of manufacture or kits herein optionally further comprise a container containing a second medicament, wherein the anti-CD70 antibody is a first medicament, and the article of manufacture or kit further comprises instructions on a label or package insert for treating a subject with an effective amount of the second medicament. In some embodiments, the label or package insert indicates that the first and second medicaments are to be administered sequentially or simultaneously.

[0229] In some embodiments, the anti-CD70 antibodies described herein are present in a container as a lyophilized powder. In some embodiments, the lyophilized powder is in a hermetically sealed container, e.g., a vial, ampule, or sachet, indicating the quantity of active agent. Where the pharmaceutical is administered by injection, an ampule of sterile water for injection or saline, for example, may optionally be provided as part of the kit so that the components can be mixed prior to administration. As would be readily apparent to one of skill in the art, such kits may optionally further comprise one or more of various conventional pharmaceutical components, e.g., containers with one or more pharmaceutically acceptable carriers, additional containers, etc. Printed instructions, either as an insert or as a label, indicating the amounts of the components to be administered, administration guidelines, and / or guidelines for mixing the components, may also be included in the kit.

[0230] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof, and should be included within the spirit and scope of this application and the appended claims. For example, the present invention encompasses the following embodiments: [Embodiment 1] A method of treating a CD70-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a nonfucosylated anti-CD70 antibody, wherein the method results in depletion of cancer cells in the subject but does not result in depletion of CD70+ regulatory T cells (CD70+ Tregs) in the subject, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising three CDRs of SEQ ID NO: 1, a light chain variable region comprising three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme, and wherein the cancer is selected from the group consisting of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). [Embodiment 2] The method described in embodiment 1, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 2. [Embodiment 3] The method described in embodiment 1, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. [Embodiment 4] The method of any one of embodiments 1 to 3, wherein the Fc domain is an antibody effector domain that mediates one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). [Embodiment 5] The method of any one of embodiments 1 to 3, wherein the Fc domain is an antibody effector domain that mediates ADCC. [Embodiment 6] The method described in any one of embodiments 1 to 5, wherein the Fc domain is a human Fc domain. [Embodiment 7] The method of any one of embodiments 1 to 6, wherein the anti-CD70 antibody is borsetuzumab. [Embodiment 8] The method of any one of embodiments 1 to 7, wherein the antibody is conjugated to a therapeutic agent. [Embodiment 9] The method of embodiment 8, wherein the therapeutic agent is a chemotherapeutic agent or an immunomodulatory agent. [Embodiment 10] The method of embodiment 8, wherein the therapeutic agent is a chemotherapeutic agent. [Embodiment 11] The method of embodiment 10, wherein the chemotherapeutic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). [Embodiment 12] The method of embodiment 8, wherein the therapeutic agent is an immunomodulatory agent. [Embodiment 13] The method of any one of embodiments 1 to 12, wherein the method comprises administering a population of anti-CD70 antibodies, each antibody in the population of anti-CD70 antibodies comprising a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibodies are defined by the Kabat numbering scheme, and at least 50% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. [Embodiment 14] The method of embodiment 13, wherein at least 70% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. [Embodiment 15] The method of embodiment 13, wherein at least 90% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation. [Embodiment 16] The method of any one of embodiments 1 to 15, wherein the cancer is MDS. [Embodiment 17] The method described in embodiment 16, wherein the MDS is relapsed or refractory MDS. [Embodiment 18] The method of embodiment 17, wherein the subject has experienced treatment failure after a previous hypomethylating agent (HMA) therapy for MDS. [Embodiment 19] The method of any one of embodiments 1 to 15, wherein the cancer is AML. [Embodiment 20] The method of embodiment 19, wherein the AML is relapsed or refractory AML. [Embodiment 21] The method of embodiment 20, wherein the subject has received two previous treatment regimens to treat AML. [Embodiment 22] The method of embodiment 20, wherein the subject has received three previous treatment regimens to treat AML. [Embodiment 23] A method described in any one of embodiments 1 to 22, wherein at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cancer cells express CD70. [Embodiment 24] The method of any one of embodiments 1 to 23, wherein administering a nonfucosylated anti-CD70 antibody to a subject results in depletion of cancer cells by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%, compared to the amount of cancer cells before administering the nonfucosylated anti-CD70 antibody to the subject. [Embodiment 25] The method of any one of embodiments 1 to 24, wherein administering a nonfucosylated anti-CD70 antibody to a subject results in depletion of CD70+ Tregs of about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about 0.1% or less compared to the amount of CD70+ Tregs before administering the defucosylated anti-CD70 antibody to the subject. [Embodiment 26] The method of any one of embodiments 1 to 25, wherein one or more therapeutic effects are improved in the subject after administration of the nonfucosylated anti-CD70 antibody relative to baseline. [Embodiment 27] The method described in embodiment 26, wherein one or more therapeutic effects are selected from the group consisting of objective response rate, duration of response, time to response, progression-free survival, and overall survival. [Embodiment 28] The method of any one of embodiments 1 to 27, wherein the objective response rate is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. [Embodiment 29] The method of any one of embodiments 1 to 28, wherein the subject exhibits a progression-free survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody. [Embodiment 30] The method of any one of embodiments 1 to 29, wherein the subject exhibits an overall survival of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody. [Embodiment 31] The method of any one of embodiments 1 to 30, wherein the duration of the response to the anti-CD70 antibody is at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years after administration of the nonfucosylated anti-CD70 antibody. [Embodiment 32] The method of any one of embodiments 1 to 31, wherein the route of administration of the anti-CD70 antibody is intravenous. [Embodiment 33] The method of any one of embodiments 1 to 32, wherein the subject is a human. [Embodiment 34] The method of any one of embodiments 1 to 33, wherein the anti-CD70 antibody is administered in combination with azacitidine. [Embodiment 35] The method of any one of embodiments 1 to 33, wherein the anti-CD70 antibody is administered in combination with venetoclax. [Embodiment 36] The method of any one of embodiments 1 to 33, wherein the anti-CD70 antibody is administered in combination with azacitidine and venetoclax. [Embodiment 37] The method of any one of embodiments 1 to 35, wherein the anti-CD70 antibody is administered in combination with a fluoroquinolone. [Embodiment 38] A pharmaceutical composition for treating a CD70-expressing cancer, comprising a nonfucosylated anti-CD70 antibody and at least one pharmaceutically compatible ingredient, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme, and the composition is for use in a method described in any one of embodiments 1 to 37. [Embodiment 39] A kit comprising a nonfucosylated anti-CD70 antibody and instructions for using the anti-CD70 antibody in a method described in any one of embodiments 1 to 37, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the three CDRs of SEQ ID NO: 1, a light chain variable region comprising the three CDRs of SEQ ID NO: 2, and an Fc domain, wherein the CDRs of the anti-CD70 antibody are defined by the Kabat numbering scheme.

[0231] [Example] [Example 1] Assessment of SEA-CD70 binding to Fcγ receptors In vivo, monocytes, macrophages, neutrophils, dendritic cells, and NK cells can mediate ADCP (antibody-dependent cell-mediated phagocytosis) and ADCC (antibody-dependent cell-mediated cytotoxicity via FcγRI, FcγRIIa, and FcγRIIIa). Although all three receptors are involved in ADCP, FcγRIIIa is thought to be the primary Fcγ receptor involved in ADCC. Non-fucosylation of IgG1 antibodies can result in higher affinity binding to FcγRIIIa and b, thus increasing ADCC and ADCP activity.

[0232] SEA-CD70 (non-fucosylated hIF6) is a humanized, non-fucosylated monoclonal antibody targeting CD70. It is being developed by Seattle Genetics for patients with refractory and / or relapsed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), for which there is currently no standard of care. SEA-CD70 is a humanized monoclonal IgG1 antibody that binds to CD70. SEA-CD70 is a non-fucosylated antibody that binds to FcγRIIIa with higher affinity than the fucosylated parent antibody SGN-70 (hIF6), and increases targeted killing of CD70-positive cells through CDC, ADCP, and amplified ADCC.

[0233] Biolayer interferometry (BLI) was used to assess the binding kinetics of SGN-70 and SEA-CD70 to FcγRI, IIa, IIIa, IIb, and FcRN. Figure 1 shows sensograms of SGN-70 (labeled as h1F6 WT) and SEA-70 (labeled as h1F6 SEA) binding to FcγRI, IIa, IIIa, IIb, and FcRN.

[0234] The binding kinetics of SGN-70 and SEA-CD70 to human FcγRI, FcγRIIa H131, FcγRIIa R131, FcγRIIIa F158, and FcγRIIIa V158 were assessed by BLI. Parameters are listed in Table 1. Biotinylated, avi-tagged human FcγR-monomeric Fc N297A LALA-PG and Fc receptor neonatal (FcRN) monomeric Fc N297A IHH fusion proteins (designed and expressed at Seattle Genetics) were loaded onto a high-precision streptavidin biosensor (ForteBio) for a response of 0.3–1 nm after a 100-second sensor check in Buffer A (0.1% bovine serum albumin [BSA], 0.02% Tween 20, 1x phosphate-buffered saline [PBS] pH 7.4). After another baseline measurement, titrated antibodies were allowed to associate for 600, 10, 100, 50, and 10 seconds, and dissociate for 1000, 50, 100, 500, and 50 seconds for FcγRI, IIa, IIIa, FcRN pH 6, and FcRN pH 7.4, respectively, in buffer B (1% casein, 0.2% Tween 20, 1x PBS pH 7.4). Reference values ​​were subtracted in each assay before analysis. All sensorgrams were processed with y-axis alignment at the onset of association and inter-step dissociation correction. Curves were fitted using a 1:1 Langmuir isotherm global fit model.

[0235] [Table 1]

[0236] Human CD70 affinity was determined by BLI using the parameters listed in Table 2. Baseline measurements in Buffer A (0.1% BSA, 0.02% Tween 20, 1x PBS pH 7.4) were taken before and after immobilization of the antibody at 6 μg / mL for 57 seconds on an AHC (anti-Fc) biosensor purchased from ForteBio. A second baseline was taken in Buffer B (1% casein, 0.2% Tween 20, 1x PBS pH 7.4), after which the titrated hCD70 analyte was allowed to associate for 600 seconds and dissociate for 1000 seconds in Buffer B. hCD70 antigen was purchased from R&D (Cat. No. 9328-CL, Lot No. DGSR0217071) and biotinylated using a 1.5-fold molar excess of EZ-Link N-hydroxysuccinimide biotin purchased from Thermo Fisher Scientific (Cat. No. 20217, Lot No. SI249775).

[0237] [Table 2]

[0238] SEA-CD70 and SGN-70 have similar on- and off-rate binding to hFcγRI and IIa. However, SEA-CD70 exhibited significantly higher binding affinity to FcγRIIIA than SGN-70. BLI experiments were performed to examine the on- and off-rates and binding affinities of SEA-CD70 and SGN-70 to FcγRI, FcγRIIa (H / H high-affinity and R / R low-affinity alleles), and FcγRIIIa (F / F low-affinity and V / V high-affinity alleles). Binding kinetics to FcRn was also performed, and SEA-CD70 and SGN-70 were found to bind with similar kinetics and affinity.

[0239] Biolayer interferometry (BLI) was used to evaluate the binding kinetics of SGN-70 and SEA-CD70 to the high-affinity FcγRIIIa(158V) receptor variant (Table 3). The nonfucosylated backbone of SEA-CD70 demonstrated an 8-fold increase in binding affinity to the FcγRIIIa(158V) receptor. Biotinylated, avi-tagged human FcγR-monomeric Fc N297A LALA-PG and FcRN-monomeric Fc N297A IHH fusion proteins (designed and expressed at Seattle Genetics) were loaded onto a high-precision streptavidin biosensor (ForteBio) for a response of 0.3–1 nm after a 200–300 s sensor check in Buffer A (0.1% BSA, 0.02% Tween 20, 1x PBS pH 7.4). After the second baseline, titrated SEA-CD70 or SGN-70 antibodies were allowed to associate until the highest concentration reached equilibrium and then dissociated until the response approached baseline. Reference values ​​were subtracted in each assay before analysis. All sensorgrams were processed with y-axis alignment at the onset of association and step-by-step dissociation correction. Curves were fitted using a 1:1 Langmuir isotherm global fit model.

[0240] [Table 3]

[0241] [Example 2] Binding of SGN-70 and SEA-CD70 to hFcγRIIIa and cFcγRIIIa by flow cytometry The BLI method is used to assess receptor affinity by monitoring binding kinetics, but is primarily designed to monitor monovalent binding. Flow cytometry was also performed to supplement the BLI dataset (Figures 2A and 2B). CHO cells were transformed to overexpress the high-affinity human FcγRIIIa receptor (158V) (Figure 2A) or the cynomolgus monkey FcγRIIIa receptor (Figure 2B) and were subjected to binding of the nonfucosylated antibody SEA-CD70 (labeled as SEA-70) or the parent fucosylated antibody SGN-70. As observed in the BLI experiments, the nonfucosylated antibody SEA-CD70 bound both human and cynomolgus monkey FcγRIIIa with higher affinity than SGN-70.

[0242] CHO-FcγRIIIa binding assays were performed as follows: 1. Thawing of cells: Cells were thawed on June 11, 2019, cultured in culture medium for one week, and recovered from freeze-thaw.

[0243] TIFF0007824220000009.tif401672. Wash: 60 million cells were washed with 1x PBS in a 50 mL tube. Cells were counted again and a concentration of 2.2 x 10 6 The cells were resuspended at 0.1 mL / mL. 0.1 mL was then pipetted per well. 3. Making 10x dilutions of antibody: 10x dilutions were prepared (3mg / mL, 1mg / mL, 0.3mg / mL, 0.1mg / mL, 0.03mg / mL, 0.01mg / mL, 0.003mg / mL, 0.001mg / mL and 0.0003mg / mL in a dilution plate).

[0244] TIFF0007824220000010.tif661684. Aspirate: Wash solution was aspirated into wells, and 100 μL of the corresponding antibody dilution was drawn with a multichannel pipette. Corresponding concentrations were 300, 100, 30, 10, 3, 1, 0.03, 0.01, 0.003, 0.001, and 0.0003 μg / mL in triplicate. Concentrations decreased vertically down the 96-well round-bottom plate. 5. Vortex: Both sides of the plate were vigorously tapped, then gently mixed using a vortex. The plate was then incubated at 4°C for 1 hour. 6. Centrifugation: Cells were centrifuged, aspirated, and washed in 200 μL of 1×BD staining buffer per well. After aspirating the final wash, cells were resuspended by vortexing the plate on a vortexer. 7. Antibody Preparation: Anti-human IgG-PE (Jackson, Cat. No. 109-116-170) was prepared by diluting 1:50 from a 1 mg / mL concentrate to a saturating concentration of 33 μg / mL. The antibody mixture was mixed thoroughly by tapping the side of the plate. The mixture was incubated in the dark in a refrigerator (4°C) for 30 minutes. 8. Washing: The mixture was centrifuged. The supernatant was then aspirated. Each well was washed with 200 μL of 2×BD staining buffer. 9. Sample Analysis: Samples were analyzed by flow cytometry in high-throughput sampler (HTS) mode on the Attune. Median fluorescence intensity (MFI) was graphed (geometric mean) and the equilibrium dissociation constant (K D ) was calculated using PRIZM.

[0245] [Example 3] ADCC of SEA-CD70 and SGN-70 in AML CD70+ cells Although SGN-70 does not directly induce apoptosis in CD70-positive target cells, SEA-CD70 mediates an effector function that may result in the elimination of target-positive cells. In a standard ADCC assay using PBMCs as a source of natural killer (NK) cells, SEA-CD70 induced the lysis of two CD70-positive AML cell lines in a dose-dependent manner, whereas lysis was not achieved with a non-binding control human IgG. These experiments demonstrated that SEA-CD70 possesses higher antibody-dependent cellular cytotoxicity than the SGN-CD70 antibody.

[0246] ADCC activity was assessed using two CD70+ AML cell lines as ADCC targets (Figures 3A and 3B). The AML cell lines, MOLM-13 (Figure 3A) and NOMO-1 (Figure 3B), were labeled and mixed with titrations of test antibodies or isotype controls. Effector cells were isolated from cryopreserved normal donor PBMCs using the EasySep Human NK Cell Enrichment Kit (Stem Cell Technologies). Effector cells were added at an effector-target cell ratio of 10:1, at 25,000:250,000. After 4 hours of incubation, specific cell lysis was calculated.

[0247] AML cell lines were grown in the appropriate growth medium while incubating at 37°C in 5% CO2. Suspension cells were counted using a VicellXR cell counter. The required volume of cells was mixed with fresh growth medium and plated at a seeding density of 0.5 M / mL.

[0248] To assess ADCC activity, the following protocol was used: 1. Two vials of huPBMCs were thawed in a 37°C water bath and resuspended in 1% FBS-RPMI medium. The cells were centrifuged, and then NK cells were isolated using the EasySep Human NK Cell Enrichment Kit according to the manufacturer's protocol. 2. Antibody titrations were performed using SGN-70 and SEA-CD70 antibodies at a starting concentration of 2 μg / mL (working concentration 6 μg / mL) and diluted 10× to 20 μg / mL in 1% FBS-RPMI medium. 3. Target tumor cells (MOLM-3 or NOMO-1) were seeded into a 96-well round-bottom plate at 50 μL / well in 1% FBS-RPMI. Antibody dilutions and isotype controls were then seeded into the same plate at 50 μL / well. Isolated NK effector cells were then seeded into the same 96-well round-bottom plate at a 1:10 tumor:NK cell ratio in 1% FBS-RPMI medium at 50 μL / well. 4. Control wells were added and the total volume was increased to 150 μL with medium. 5. The test plate was incubated for 4 hours at 37°C in a 5% CO2 incubator. After 45 minutes of incubation, 15 μL / well of lysis solution was added to the Max lysis control wells and returned to the incubator for the remainder of the 4 hour incubation. 6. The test plate was centrifuged at 250 xg for 4 minutes and 50 μL of supernatant from each well was transferred to a new flat-bottom clear plate. 7. CytoTox 96 reagent was added at 50 μL / well and incubated in the dark for 30 minutes at room temperature. Stop solution was then added to all wells at 50 μL / well. 8. The absorbance per well was measured at 490 nm using a SpectraMax 190 plate reader and the resulting values ​​were converted into a text file and exported to Excel and GraphPad Prism for further data analysis. 9. Cytotoxicity is reported as the maximum percentage of lysis achieved by lysis solution treatment, subtracting background.

[0249] [Example 4] The effects of SGN-70 and SEA-CD70 on regulatory T cells To assess the effect of SEA-CD70 on CD70+ T cell depletion, PBMCs containing naive, memory, and Treg subsets were treated with increasing concentrations of SEA-CD70 or SEA-CD70 for 24 hours. At the end of the experiment, cells were stained with Zombie Aqua Viability Dye to assess the total number of viable Treg naive and memory CD4+ and CD8+ T cells. Depletion assessments were performed using donors homozygous for the low-affinity FcγRIIIa receptor (F / F 158) (Figures 4C and 4D) or homozygous for the high-affinity FcγRIIIa receptor (V / V158) (Figures 4A and 4B). Fucosylated (WT clone 13IgG1) and nonfucosylated (SEA clone 13IgG1) antibodies targeting TIGIT were used as positive controls (Figures 4E–4H). Neither CD70-targeting antibody induced T regulatory cell depletion in the low-affinity F / F158 donor (Figure 4C). Fucosylated anti-CD70 antibody (labeled as SGN-CD70) resulted in T regulatory cell depletion when V / V high-affinity donors were used, whereas, surprisingly, the nonfucosylated antibody SEA-CD70 increased ADCC activity in CD70+ AML cell lines but did not induce Treg cell depletion (Figure 4A). Neither fucosylated nor nonfucosylated anti-CD70 antibody depleted CD8+ cells, regardless of whether V / V high-affinity donors (Figure 4B) or low-affinity donors (Figure 4D) were used.

[0250] In contrast to what was observed with the CD70-targeted antibody, the nonfucosylated anti-TIGIT antibody (labeled as SEA clone 13 IgG1) depleted Treg cells to a greater extent than the fucosylated anti-TIGIT antibody (labeled as WT clone 13 IgG1) when using either the V / V high-affinity donor (Figure 4E) or the low-affinity donor (Figure 4G).

[0251] The lack of Treg depletion by SEA-CD70 compared to SGN-70 is surprising not only in light of the results comparing fucosylated and nonfucosylated anti-TIGIT antibodies, but also in light of publications reporting the activity of other nonfucosylated antibodies. For example, U.S. Patent Application Publication No. 2019 / 0284287 shows that a nonfucosylated anti-CD25 antibody has greater ADCC activity than a corresponding fucosylated anti-CD25 antibody, resulting in greater lysis and depletion of induced Tregs (iTregs). Similarly, U.S. Patent Application Publication No. 10,196,445 shows that a nonfucosylated anti-CTLA4 antibody, but not a corresponding fucosylated anti-CTLA4 antibody, results in Treg lysis and depletion.

[0252] It has been shown that depletion of Tregs can have negative, even fatal, consequences. For example, depletion of Tregs using diphtheria toxin has been shown to cause severe autoimmune disorders in two mouse models. See Kim et al. (2009) J. Immunol. 183:7631-7634. ​​Furthermore, acute ablation of Treg cell populations can lead to end-stage autoimmune disease. See Kim et al. (2007) Nat. Immunol. 8(2):191-7.

[0253] [Example 5] Phase I Clinical Trial of SEA-CD70 in Patients with Myeloid Malignancies This is a phase 1, open-label, multicenter, dose-escalation and cohort expansion study designed to evaluate the safety, tolerability, pharmacokinetics (PK), and antitumor activity of SEA-CD70 in adults with myeloid malignancies. The safety and efficacy of SEA-CD70 in patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), are evaluated here. The study will assess what side effects occur and whether SEA-CD70 is an effective treatment for MDS and AML.

[0254] This study has three parts and enrolled a total of 60 subjects. Part A is a dose-escalation cohort designed to identify the maximum tolerated dose (MTD) or recommended dose escalation of SEA-CD70 monotherapy in subjects with relapsed / refractory MDS, e.g., after treatment failure with hypomethylating agents (HMA-failure). Part B is an expansion cohort designed to evaluate the safety and tolerability of SEA-CD70 monotherapy in subjects with relapsed / refractory MDS, e.g., after HMA-failure. Part C is an expansion cohort designed to evaluate the safety and tolerability of SEA-CD70 monotherapy in subjects with relapsed / refractory AML. Subjects enrolled in this study were aged 18 years or older and included both male and female subjects. SEA-CD70 was administered on days 1 and 15 of each treatment cycle. All treatment components were administered intravenously. The inclusion and exclusion criteria for subjects enrolled in this study are shown in Table 4.

[0255] [Table 4] TIFF0007824220000012.tif245169TIFF0007824220000013.tif162170

[0256] The endpoints are listed in Table 5. All treatment components are administered intravenously.

[0257] [Table 5] TIFF0007824220000015.tif121167

[0258] [Example 6] Dose-dependent effect of h1F6SEA on survival in the Raji NHL Burkitt lymphoma mouse model. Studies have shown that acute myeloid leukemia (AML) and myelodysplastic diseases (MDS) express CD70 and its receptor CD27. The purpose of this study was to examine animal survival in response to the anti-CD70 monoclonal antibody SEA-CD70 (h1F6SEA). Animal survival in response to administration of SEA-CD70 was evaluated in a CD70-expressing cell xenograft mouse model, the Raji NHL-Burkitt model.

[0259] SCID mice were injected with 1 × 10 6 Raji cells were implanted intravenously. Animals were randomly assigned to treatment groups with eight mice per group on day 1 post-implantation. Animals were intraperitoneally dosed with h1F6SEA at 0.3, 1, and 3 mg / kg once every four days (Q4dx4) starting on day 1 post-tumor implantation. Stock-concentration antibody was diluted to the appropriate concentration and injected into animals at 10 μl / g body weight. Animals were then monitored for disease symptoms. Animals were followed until disease symptoms appeared and then euthanized. Animals were analyzed over time, and animals were sacrificed if disease symptoms were observed. Animals in the untreated group had a median survival time of 20 days, while animals treated with 0.3 mg / kg h1F6SEA progressed to 36.5 days, and animals treated with 1 or 3 mg / kg progressed to 68 and 69.5 days post-implantation. The total number of animals in each group on each day during the study is shown in Table 6. Survival rates were calculated for animals in all treatment groups (Figure 5). Kaplan-Meyer graphs show a significant increase in survival rate between treated and untreated animals, and a dose response between 0.3 mg / kg and 1 or 3 mg / kg (Figure 5). Survival rates were quantified across experimental days for all treatment groups, including h1F6SEA doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Treatment of mice with h1F6SEA increased survival compared to untreated mice (Figure 5).

[0260] [Table 6]

[0261] [Example 7] Dose-dependent effect of h1F6SEA on tumor growth in the MV411 acute myeloid leukemia mouse model. In this study, tumor growth in response to administration of the anti-CD70 antibody SEA-CD70 (h1F6SEA) was evaluated in a mouse model of acute myeloid leukemia, a CD70-expressing cell xenograft of the MV-411 lineage. Tumor growth was reported as volume and calculated as the average across animals within each treatment group (Figure 6), as well as for each individual within each treatment group (Figures 7A-D, Table 7). Daily tumor volumes (mm) from individual animals within the different treatment groups were calculated. 3 ) are summarized in Table 7.

[0262] SCID mice were injected subcutaneously in the flank on day 0 with 5 × 10 6 MV-411 cells were transplanted. The average tumor diameter was 50 mm. 3 (Formula: Volume (mm 3 ) = 0.5 x length x width 2 When tumor size reached 1000 mm (measured using a 1000 μg / kg / day RT-PCR) (length being the longer dimension), mice were randomly assigned to treatment groups with six animals per group. Animals were treated according to treatment group; groups receiving antibodies were treated every four days for four cycles, and animals receiving azacitidine were treated every four days for four cycles. Treatments were given intraperitoneally. Stock concentrations of antibodies and chemotherapy were diluted to the appropriate concentrations and injected into animals at 10 μl / g body weight. During the study, tumor length and width, as well as animal weights, were measured twice weekly, and tumor volume was calculated using the formula above. Approximately 1000 mm 3Animals were followed until tumor volumes of 1000 mg / kg were measured, at which point they were euthanized. Animals were dosed on various schedules based on the treatment they received 9 days after tumor implantation: animals receiving antibodies were treated with Q4dx4, and animals treated with azacytidine were dosed once daily every 4 days for a total of four cycles (Q4dx4). Analysis of tumor volume change over time showed a slight tumor delay compared to animals treated with untreated or non-conjugated antibody (Figures 6 and 7A-D). When examining the time it took for tumors in each group to reach a 10-fold change, the untreated group took an average of 26.8 days, while the h1F6SEA 10 mg / kg-treated group took an average of 32.65 days, representing an 18% delay in tumor growth (Figures 6 and 7A-D). However, this could have been longer, as one animal did not reach a 10-fold change. Animals treated with azacitidine (labeled as Vidaza in Figures 6, 7, and Table 7) also showed a growth delay that required 33.68 days to reach a 10-fold change, representing a 20.5% delay in tumor growth (Figures 6 and 7A-D). It should also be noted that one mouse in the h1F6SEA 10 mg / kg group showed very robust tumor growth delay that extended the length of the study (Figure 7A and Table 7).

[0263] [Table 7]

[0264] [Example 8] Evaluation of SEA-CD70 and SGN-CD70-mediated ADCP activity against AML cell lines. SEA-CD70- and SGN-CD70-(also called SGN-70)-mediated ADCP was determined using CD70+ target cells (NOMO-1 and MOLM-13) loaded with a lipophilic fluorescent dye and mixed overnight with monocyte-derived macrophages. Phagocytosis of fluorescently labeled target cells was determined by flow cytometry. Phagocytosis was measured for the appearance of double-labeled coincidence events (fluorescent target cells) and anti-CD11c positivity to identify monocytes / macrophages. Macrophages readily engulfed target cells coated with either SEA-CD70 or SGN-CD70 in an antibody dose-dependent manner (Figures 8A and 8B). SEA-CD70 and SGN-CD70 mediated similar levels of phagocytosis.

[0265] The following protocol was used to assess ADCC activity in AML cell lines: 1. Target cells were labeled using the PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma Aldrich) according to the manufacturer's instructions. 2. Cells (4000 cells / well) were incubated with the indicated test substances for 30 minutes, washed and resuspended in RPMI + 10% ultra-low IgG FBS. 3. PBMC-derived macrophages (100,000 cells / well) generated by incubating PBMC-derived monocytes with 500 U / mL (50 ng / mL) GM-CSF for 10–12 days were added to the target cells and incubated at 37°C for 2 hours. 4. The plates were centrifuged and the cells were resuspended in 100 μl of APC-CD11 antibody (macrophage marker) and incubated on ice for 30 minutes. 5. Cells were washed, resuspended in PBS and analyzed by flow cytometry to determine the percentage of phagocytosis.

[0266] [Example 9] Evaluation of SEA-CD70- and SGN-CD70-mediated CDC activity against AML cell lines. The ability of SEA-CD70 and SGN-CD70 to induce cell lysis via complement fixation was further tested. CD70-positive AML cell lines were fluorescently labeled and treated with increasing concentrations of CD70-directed antibodies. The cells were then exposed to human complement, and lysis was determined as fluorescent dye release. The CD70+ AML cell lines MOLM-13 and NOMO-1 were lysed in an antibody-specific and dose-dependent manner when coated with either SEA-CD70 or SGN-CD70 in the presence of non-heat-inactivated normal human serum (Figures 9A and 9B).

[0267] The following protocol was used to assess CDC activity in AML cell lines: 1. Cells were incubated with 10 mg / ml anti-CRP monoclonal antibody mixture (anti-hCD46, anti-hCD55, anti-hCD59) on ice for 30 minutes. 2. Cells were washed and plated (200,000 cells / well) in medium containing non-heat-inactivated serum, Sytox Green (Life technologies), and antibodies at the indicated final concentrations for 2 hours at 37°C. 3. Cell death was quantified by detecting the Sitox green fluorescent signal on an Envison plate reader (Perkin Elmer) and normalized to a positive control (1% Triton X-100 treated cells).

[0268] [Example 10] Effect of the combination of SEA-CD70 and azacytidine on tumor growth in the MV411 AML xenograft mouse model In this study, tumor growth in response to administration of the defucosylated anti-CD70 antibody SEA-CD70 (h1F6SEA) alone or in combination with azacytidine (VIDAZA®) was evaluated in a CD70-expressing cell xenograft mouse model, MV4-11 line. Tumor growth was reported as volume and calculated as the average across animals within each treatment group (Figure 10). SCID mice were implanted with 5x10e6 MV4-11 cells subcutaneously in the flank on day 0. The mean tumor diameter was 50mm. 3 (Formula: Volume (mm 3 ) = 0.5 x length x width2 When tumor volume reached approximately 1000 mm (measured using the formula (x, y ... 3Animals were followed until tumor volumes of 1000 mg / kg were measured, at which point they were euthanized. Animals were dosed on various schedules based on the treatment they received: animals receiving antibodies were treated at a dose of 10 mg / kg (Q4dx5); animals treated with azacitidine were dosed once daily for five consecutive days for a total of three cycles (three weeks) (azacytidine 2 mg / kg; Q1dx5); and animals receiving the treatment combination received each treatment at the same dose and schedule as the single treatments. Analysis of tumor volume change over time indicates that both azacitidine and SEA-CD70 reduce tumor growth when compared to control, untreated animals. Furthermore, when animals were treated with the combination of SEA-CD70 and azacitidine, a further increase in tumor delay was observed compared to untreated animals and both single SEA-CD70 or azacitidine treatments (Figure 10). As expected, treatment with the SEA-CD70 G1V1 antibody, which has Fc domain mutations (E233P, L234V, L235A) that reduce binding to Fc gamma receptors (see McEarchern et al., 2008, Clin. Cancer Res. 14(23):7763-72; Armour et al., 1999, Eur. J. Immunol. 29:2613-2624), was ineffective in reducing tumor growth. Surprisingly, when SEA-CD70 G1V1 was combined with azacitidine, a significant delay in tumor growth was observed (Figure 10). Without wishing to be bound by any theory, the underlying mechanism of this observation may be related to changes in CD70 or CD27 expression or inhibition of CD27 / CD70 signaling caused by azacitidine treatment. When examining the time it took for tumors in each group to reach a 10-fold change, untreated animals took an average of 17.82 days, while the SEA-CD70-treated group took an average of 25.08 days, representing a 31% delay in tumor growth (Figure 10). Animals treated with azacitidine also showed a growth delay of 26.59 days to reach a 10-fold change, representing a 33% delay in tumor growth compared to untreated controls (Figure 10).Animals treated with the combination of azacitidine and SEA-CD70 took an average of 33.81 days (47.3% delay in tumor growth) to reach a 10-fold increase (Figure 10), demonstrating that the combination of SEA-CD70 and azacitidine effectively delayed tumor growth compared with the two drugs used as single agents.

[0269] [Example 11] Effect of SEA-CD70 in combination with azacitidine, venetoclax (ABT-199), or both (azacitidine + venetoclax) on tumor growth in the MV4-11 AML xenograft mouse model. In this study, tumor growth in response to administration of the defucosylated anti-CD70 antibody SEA-CD70 (h1F6SEA) alone or in combination with azacitidine (VIDAZA®), venetoclax (VENCLEXTA®; ABT-199), or SEA-CD70 + azacitidine + venetoclax (triplet combination) was evaluated in the CD70-expressing cell xenograft mouse model MV4-11. Tumor growth was reported as volume and calculated as the average across animals within each treatment group (Figure 11A). Immunodeficient SCID mice were implanted subcutaneously in the flank with 5x10e6 MV4-11 cells on day 0. A mean tumor diameter of 50 mm was observed. 3 (Formula: Volume (mm 3 ) = 0.5 x length x width 2 When tumor volume reached approximately 1000 mm (measured using the formula (length is the longer dimension)), mice were randomly assigned to treatment groups of 10 mice per group. Stock concentrations of antibodies and chemotherapy were diluted to the appropriate concentrations and injected into the animals at 10 μl / g body weight. During the study, tumor length and width, as well as animal weight, were measured twice weekly, and tumor volume was calculated using the formula above. 3Animals were followed until tumor volumes of 0.001 mg / kg were measured, at which point the animals were euthanized. Animals were dosed on various schedules based on the treatment they received: animals receiving antibody were treated with 10 mg / kg antibody (Q4dx5); animals treated with azacitidine were dosed daily (2 mg / kg) for 5 consecutive days (Q1dx5) every week for a total of 3 cycles (3 weeks); venetoclax was given by oral gavage at 25 mg / kg daily for 21 consecutive days (Q1dx21). Animals receiving treatment combinations received each treatment at the same dose and schedule as the single treatments.

[0270] As shown in Figure 11A, SEA-CD70, azacitidine, and venetoclax treatments slow tumor growth when administered as single agents. Notably, the addition of SEA-CD70 to either azacitidine or venetoclax significantly reduced tumor growth compared to the relative single-arm treatments (p<0.05 for both comparisons on day 39, two-way ANOVA). Furthermore, the combination of venetoclax and azacitidine further inhibited tumor growth compared to the single agents (p<0.01 and p<0.001, respectively, when compared to azacitidine or venetoclax single arms on day 39, two-way ANOVA).

[0271] Adding SEA-CD70 to venetoclax and azacitidine (triplet combination) further delayed tumor growth compared with the two-drug combination (p=0.0594 at day 39, two-way ANOVA). At day 46, the azacitidine + venetoclax combination group had a mean tumor diameter of 349.9±141.7 mm 3 (mean ± SEM), whereas the triplet combination resulted in a mean tumor diameter of 85 ± 11.09 mm. 3 (mean ± SEM) (p<0.05; one-tailed t-test). As shown in Figure 11B, when observing single animal tumor growth curves, at day 56, five animals treated with the venetoclax + azacytidine combination reached a 10-fold increase in tumor volume, while only one animal treated with the triplet combination reached the 10-fold threshold at which time the experiment was discontinued.

[0272] Overall, these results showed that tumor growth was further delayed when SEA-CD70 was added to standard treatment (azacitidine, venetoclax, or the combination of azacitidine and venetoclax).

Claims

1. 1. A composition comprising a therapeutically effective amount of a nonfucosylated anti-CD70 antibody for use in a method of treating a CD70-expressing cancer in a subject, said method resulting in depletion of cancer cells in the subject and resulting in depletion of CD70 regulatory T cells (CD70 Tregs) by 20% or less compared to the amount of CD70 Tregs before administering the nonfucosylated anti-CD70 antibody to the subject, wherein the CDRs of the anti-CD70 antibody are numbered according to the Kabat numbering scheme. a heavy chain variable region comprising heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3) in SEQ ID NO: 1, a light chain variable region comprising light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), and light chain CDR3 (L-CDR3) in SEQ ID NO: 2, and an Fc domain, wherein the cancer is selected from the group consisting of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

2. 2. The composition of claim 1, wherein the anti-CD70 antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

2.

3. The composition of claim 1 , wherein the anti-CD70 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

4. 4. The composition of claim 1, wherein the Fc domain is an antibody effector domain that mediates one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

5. The composition of claim 1 , wherein the Fc domain is an antibody effector domain that mediates ADCC.

6. The composition of claim 1 , wherein the Fc domain is a human Fc domain.

7. The composition of any one of claims 1 to 6, wherein the anti-CD70 antibody is borsetuzumab.

8. The composition of claim 1 , wherein the antibody is conjugated to a therapeutic agent.

9. The composition of claim 8 , wherein the therapeutic agent is a chemotherapeutic agent or an immunomodulatory agent.

10. The composition of claim 8 , wherein the therapeutic agent is a chemotherapeutic agent.

11. 11. The composition of claim 10, wherein the chemotherapeutic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

12. The composition of claim 8 , wherein the therapeutic agent is an immunomodulatory agent.

13. 13. The composition of any one of claims 1 to 12, wherein the method comprises administering a population of anti-CD70 antibodies, wherein each antibody in the population of anti-CD70 antibodies comprises a heavy chain variable region comprising heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3) of SEQ ID NO: 1, a light chain variable region comprising light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), and light chain CDR3 (L-CDR3) of SEQ ID NO: 2, wherein the CDRs of the anti-CD70 antibodies are defined according to the Kabat numbering scheme, and an Fc domain, wherein at least 50% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation.

14. The composition of claim 13, wherein at least 70% of the anti-CD70 antibodies in the population of anti-CD70 antibodies lack core fucosylation.

15. 15. The composition of any one of claims 1 to 14, wherein the cancer is MDS.

16. 16. The composition of claim 15, wherein the MDS is relapsed or refractory MDS.

17. 17. The composition of claim 16, wherein the subject has experienced treatment failure after a previous hypomethylating agent (HMA) therapy for MDS.

18. The composition of any one of claims 1 to 14, wherein the cancer is AML.

19. 19. The composition of claim 18, wherein the AML is relapsed or refractory AML.

20. 20. The composition of claim 19, wherein the subject has received two prior treatment regimens to treat the AML.

21. 20. The composition of claim 19, wherein the subject has received three prior treatment regimens to treat the AML.

22. 22. The composition of any one of claims 1 to 21, wherein at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cancer cells express CD70.

23. 23. The composition of any one of claims 1 to 22, wherein administering the composition to a subject results in at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% depletion of cancer cells compared to the amount of cancer cells before administering the nonfucosylated anti-CD70 antibody to the subject.

24. 24. The composition of any one of claims 1 to 23, wherein administering the composition to a subject results in depletion of CD70+ Tregs by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% compared to the amount of CD70+ Tregs before administering the nonfucosylated anti-CD70 antibody to the subject.

25. 25. The composition of any one of claims 1 to 24, wherein one or more therapeutic effects are improved in a subject following administration of the nonfucosylated anti-CD70 antibody relative to baseline.

26. 26. The composition of claim 25, wherein the one or more therapeutic effects are selected from the group consisting of objective response rate, duration of response, time to response, progression-free survival, and overall survival.

27. 27. The composition of any one of claims 1 to 26, wherein the route of administration is intravenous.

28. 28. The composition of any one of claims 1 to 27, wherein the subject is a human.

29. (i) the anti-CD70 antibody is administered in combination with azacitidine; (ii) the anti-CD70 antibody is administered in combination with venetoclax; (iii) the anti-CD70 antibody is administered in combination with azacitidine and venetoclax; (iv) the anti-CD70 antibody is administered in combination with a fluoroquinolone; (v) the anti-CD70 antibody is administered in combination with azacitidine and a fluoroquinolone; (vi) the anti-CD70 antibody is administered in combination with venetoclax and a fluoroquinolone; or (vii) the anti-CD70 antibody is administered in combination with azacitidine, venetoclax, and a fluoroquinolone; 29. The composition of any one of claims 1 to 28.

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