Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/035—Animal model for multifactorial diseases
  • A01K2267/0387—Animal model for diseases of the immune system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/72—Increased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention relates generally to antibodies against cytosolic proteins. More particularly, the invention relates to antibodies against Wilm's tumor oncogene protein (WT1 ), specifically antibodies that recognize a WT1 peptide in conjunction with a major histocompatibility antigen.
• WT1 Wilm's tumor oncogene protein
• Therapeutic monoclonal antibodies are highly specific and effective drugs, with pharmacokinetics suitable for infrequent dosing.
• all current marketed therapeutic anticancer mAbs target extracellular or cell-surface molecules, whereas many of the most important tumor-associated and oncogenic proteins are nuclear or cytoplasmic (Sensi M and Anichini A. Clinical cancer research: an official journal of the American Association for Cancer Research 2006;12(17):5023- 32; Kessler JH and Melief CJ. Leukemia 2007;21 (9):1859-74).
• Intracellular proteins are processed by the proteasome and presented on the cell surface as small peptides in the pocket of major histocompatibility complex (MHC) class I molecules (in humans, also called human leukocyte antigen, HLA) allowing recognition by T-cell receptors (TCRs) (Morris E et al. Blood Rev 2006;20(2):61 -9; Konig R. Curr Opin Immunol 2002;14(1 ):75-83). Therefore, mAbs that mimic the specificity of TCRs (that is, recognizing a peptide presented in the context of a specific HLA-type) can bind cell-surface complexes with specificity for an intracellular protein.
• MHC major histocompatibility complex
• HLA human leukocyte antigen
• TCRm TCR-mimic antibody
• Andersen PS et al. Proceedings of the National Academy of Sciences of the United States of America 1996;93(5):1820-4 A "TCR-mimic” (TCRm) antibody was first reported by Andersen et. al. (Andersen PS et al. Proceedings of the National Academy of Sciences of the United States of America 1996;93(5):1820-4), and several have since been developed by various groups (Epel M et al. European journal of immunology 2008;38(6):1706-20; Wittman VP et al. J Immunol 2006;177(6):4187-95; Klechevsky et al. Cancer research 2008;68(15):6360-7; Bhattacharya R et al. Journal of cellular physiology 2010;225(3):664-72; Verma B, et al. J Immunol 2010;184(4):2156-65; Sergeeva et al. Blood 201 1 ;1 17(16):4262-72).
• ESK1 The first fully human TCRm mAb, called ESK1 , that specifically targets RMFPNAPYL (RMF), a peptide derived from Wilms' tumor gene 1 (WT1 ), presented in the context of HLA-A0201 was recently reported (Dao T et al. Science translational medicine 2013;5(176):176ra33). WT1 is an important, immunologically validated oncogenic target that has been the focus of many vaccine trials (Dao T et al. Best practice & research Clinical haematology 2008;21 (3):391 -404).
• WT1 is a zinc finger transcription factor with limited expression in normal adult tissues, but is over expressed in the majority of leukemias and a wide range of solid tumors (Sugiyama H. Japanese journal of clinical oncology 2010;40(5):377-87). WT1 was ranked as the top cancer antigenic target for immunotherapy by a National Institutes of Health-convened panel (Cheever MA et al. Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15(17):5323-37); further, WT1 expression is a biomarker and a prognostic indicator in leukemia (Inoue K et al. Blood 1994;84(9):3071 - 9, Ogawa H et al.
• ESK1 mAb specifically bound to leukemias and solid tumor cell lines that are both WT1 + and HLA-A0201 + and showed efficacy in mouse models in vivo against several WT1 + HLA-A0201 + leukemias (Dao T et al. Science translational medicine 2013;5(176):176ra33). Therefore, ESK1 is a useful therapeutic platform for further clinical development, and improvements to the native antibody could help potentiate its effect and improve clinical efficacy.
• the invention relates to an antibody comprising: (A) a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; 18, 19 and 20; 34, 35, and 36; 50, 51 , and 52; 66, 67, and 68 or 82, 83, and 84; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; 24, 25 and 26; 40, 41 and 42; 56, 57 and 58; 72, 73 and 74 or 88, 89 and 90; or (B) a VH and VL comprising the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 16; 30 and 32; 46 and 48; 62 and 64; 78 and 80 or 94 and 96,
• the altered antibody exhibits between 50-100% (80%) higher affinity for activating human FcYRIIIa (158V variant) than normally glycosylated antibody, has 3-4-fold (3.5-fold) higher affinity for a FcYRIIIa 158F variant than normally glycosylated antibody, and has between 30 and 70% (50%) reduced affinity for inhibitory FcyRllb than normally glycosylated antibody.
• the antibody comprises a light chain consisting essentially of the amino acid sequence of SEQ ID NO: 100 and a heavy chain consisting essentially of the amino acid sequence of SEQ I D NO: 101 .
• the invention relates to isolated nucleic acids, vectors and cells comprising a nucleic acid that encodes an antibody as described herein.
• the invention relates to the use of an antibody as disclosed herein for the treatment of a WT1 positive disease and therefore, to a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
• the invention relates to a method for treatment of a subject having a WT1 -positive disease, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein.
• WT1 -positive disease amenable to treatment with the antibody of the invention includes chronic leukemia or acute leukemia or WT1 + cancer, including, for example, chronic myelocytic leukemia, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma.
• MM multiple myeloma
• ALL acute lymphoblastic leukemia
• AML acute myeloid/myelogenous leukemia
• MDS myelodysplastic syndrome
• Figure 1 shows that ESKM has a modified Fc glycosylation pattern, altering binding to FcyRs but not to the RMF/A2 target.
• (1A) Comparison of the oligosaccharide profile of ESK1 and ESKM. Peak assignment is based on the retention time and the monosaccharide composition analysis. G# indicates the number of terminal galactoses (0, 1 , or 2), F indicates presence of core fucose, Hex5GlcNAc2 denotes (GlcNAc)2 core with terminal Hexose 5 glycan structure (terminating in mannose and/or glucose).
• Anti-mouse FcR binding was assessed by ELISA, while anti-human FcR binding was determined by FCM titration on FcyR-expressing CHO cells. Representative binding curves of ESK1 and ESKM against human FcRn (1 C), mouse FcyRIV (1 D), and mouse FcyRllb (1 E). 125 l-labeled ESK1 (1 F) and ESKM (1G) mAbs were titrated against JMN cells. All curves were fit with a non-linear single-site total binding saturation curve, and K d was calculated using Prism software.
• ESKM having 100% reduced fucose content relative to ESK1 wildtype lgG1 showed improved reverse signaling through FcyRllla compared to ESK1 wildtype lgG1 and ESK1 containing D265A/P329A mutations in the Fc domain (ESK1 -DAPA) (1 H).
• FIG. 2 shows that ESKM is more efficacious and potent in ADCC assays with human PBMC effectors at the indicated mAb concentrations and effector/target (E:T) ratios. Cytotoxicity was measured by 4-hour 51 Cr release assay.
• (2A) T2 cells were pulsed with RMF peptide and incubated with 3 pg/mL mAb.
• HLA-A0201 + human mesothelioma cell lines (2F) JMN, (2G) Meso37 and (2H) Meso56. Data presented are averages of triplicate measurements from representative experiments, all with isolated PBMCs from the same donor. All cell lines, with exception of Meso37 and Meso56, were repeated 3 or more times with multiple donors.
• FIG. 3 shows that ESKM more effectively treats JMN mesothelioma in SCID mice.
• 3D ESKM is effective against SET2 AML ( * p ⁇ 0.05, multiple T-tests).
• 3E-3F ESKM is effective against a disseminated fresh, patient-derived pre-B ALL ( * p ⁇ 0.05, ** p ⁇ 0.01 , multiple T-tests).
• Bone marrow cells from the isotype-treated mice were injected into the right shoulder flank (viewed from above), while an equal number of bone marrow cells from the ESKM-treated mice were injected into the left shoulder.
• 4C Pharmacokinetics of ESKM (2 g) in C57BL6/J or HLA-A0201 + transgenic mice.
• 4D Biodistribution of ESKM (100 pg) in C57BL6/J or HLA-A0201 + transgenic mice, harvested after 1 day.
• FIG. 5 shows that ESKM treatment does not affect leukocyte or hematopoietic stem cell (HSC) counts in HLA-A0201 + transgenic mice.
• (5A) Total white blood cell (WBC) and WBC subset cell counts. Absolute number (5B) and frequency (5C) of lineage- SCA1 + KIT+ (LSK) cells. Absolute number (5D) and frequency (5E) of long-term HSCs (Slarnf CD34- LSK cells).
• Figure 6 shows that ESKM has no significant effect against intraperitoneal JMN mesothelioma in NOG mice.
• Mice were engrafted intraperitoneally with 3x10 6 luciferase+ JMN cells, then treated with 50 ⁇ g ESKM or hlgG1 isotype control antibody twice weekly starting on day 4 via intraperitoneal injections.
• Figure 7 shows that all human antibodies tested accumulated more in spleens of HLA-A2+ transgenic mice, but ESK1 did not bind specifically to isolated HLA- A2+ spleen, bone marrow or thymus cells.
• (7A) Accumulation of 125 l-labeled antibodies in spleens of C57BL6/J or HLA-A2+ transgenic mice relative to antibody level in the blood. Mice were injected retroorbitally with 2 g indicated antibody, then sacrificed after 24 hours for blood and spleen collection.
• ADCC Antibody-dependent cellular cytotoxicity
• ALL Acute lymphocytic leukemia
• AML Acute myeloid leukemia
• CMC Complement mediated cytotoxicity
• CDR Complementarity determining region (see also HVR below)
• CH1 1 st constant domain of the heavy chain
• CH2 2nd and 3rd constant domains of the heavy chain
• CHO Chinese hamster ovary
• FACS Flow assisted cytometric cell sorting
• FBS Fetal bovine serum
• HLA Human leukocyte antigen
• HVR-H Hypervariable region-heavy chain (see also CDR)
• HVR-L Hypervariable region-light chain (see also CDR)
• IRES Internal ribosome entry site
• MHC Major histocompatibility complex
• MM Multiple myeloma
• VH Variable heavy chain includes heavy chain hypervariable region and heavy chain variable framework region
• VL Variable light chain includes light chain hypervariable region and light chain variable framework region
• WT1 Wilms tumor protein 1
• administering and “administration” refer to the
• Antibody and “antibodies” as those terms are known in the art refer to antigen binding proteins of the immune system.
• the term “antibody” as referred to herein includes whole, full length antibodies having an antigen-binding region, and any fragment thereof in which the "antigen-binding portion” or “antigen-binding region” is retained, or single chains, for example, single chain variable fragment (scFv), thereof.
• a naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region.
• the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region.
• the light chain constant region is comprised of one domain, CL.
• the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• Each VH and VL is composed of three CDRs and four FRs arranged from amino- terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
• the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical complement system.
• antigen-binding portion or "antigen-binding region” of an antibody, as used herein, refers to that region or portion of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen- binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an peptide/HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments
• antibody fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 1989;341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).
• Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
• F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
• a Fd fragment consisting of the VH and CH1 domains
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
• scFv single chain Fv
• the term "effective amount” means that amount of a compound or therapeutic agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
• terapéuticaally effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
• the term also includes within its scope amounts effective to enhance normal physiological function.
• TCRm antibodies are potentially limited by the extremely low number of epitopes presented on the target cell, which may be as few as several hundred sites (Dao T et al. Science translational medicine 2013;5(176):176ra33). Therefore, mechanisms to enhance potency may be essential to their success in humans as therapeutic agents against cancer.
• the mechanisms of action of mAbs can be enhanced through Fc region protein engineering (Desjarlais JR et al. Drug discovery today 2007;12(21 -22):898-910), or by modification of Fc-region glycosylation (Jefferis R. Biotechnology progress 2005;21 (1 ):1 1 -6; Hodoniczky J et al. Biotechnology progress 2005;21 (6):1644-52). Removal of fucose from the carbohydrate chain increases mAb binding affinity for the activating FcvRllla receptor and enhances ADCC (de Romeuf C et al. British journal of haematology 2008;140(6):635-43; Masuda K, et al.
• An Fc-modified antibody can be generated by expressing a construct encoding an anti-WT1 /HLA/A2 antibody, for example, as disclosed in WO 2012/135854, in MAGE 1 .5 CHO cells in accordance with methodology disclosed in U.S. 8,025,879 (Eureka Therapeutics, Inc), resulting in a consistent pattern of defucosylation and exposed terminal hexose (mannose and/or glucose), allowing higher affinity for activating human FcvRllla and murine FcyRIV while decreasing affinity for inhibitory FcyRllb.
• a modified antibody of the present disclosure designated herein as "ESKM" has reduced fucose content and/or galactose content, e.g., relative to a wild-type antibody.
• the fucose content and/or galactose content can be reduced by 30% to 100% using any method known in the art.
• HSCs hematopoietic stem cells
• the antibody of the invention is an anti-WT1/HLA-A2 antibody having an antigen binding region that specifically binds to a WT1 peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1 ) in conjunction with HLA-A0201 .
• the antibody of the invention comprises one of the combinations of amino acid sequences for CDRs, and heavy and light chain variable regions from Tables 1 -6.
• the light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region.
• the heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region.
• the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.
• light and heavy chain constant regions may have the amino acid sequences (as shown in Table 7) of SEQ ID NO. 98 and SEQ ID NO. 99, respectively.
• the antibody of the invention comprises a light chain and heavy chain with amino acid sequences as follows:
• a leader sequence, MGWSCI I LFLVATATG (SEQ ID NO: 102), as shown in gray may be included.
• CDRs are bolded in Table 8 and correspond to the CDRs listed in Table 3.
• nucleic acid that encodes for the variable and hypervariable (CDR) regions of the heavy or light chain is also shown.
• Vectors and other nucleic acid constructs which comprise a nucleic acid that encodes the antibody and which can be used for expression of the antibodies from MAGE 1 .5 CHO cells are also encompassed by the invention.
• the antibodies of the present disclosure also include substantially homologous polypeptides having antigen-binding portions that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the peptides described in Tables 1 - 6 or 8.
• an antibody of the present disclosure comprises a heavy chain variable region comprising CDR1 , CDR2, and CDR3 from a VH sequence in any of Tables 1 -6 or 8 that is at least 90% identical to that VH sequence and/or comprises a light chain variable region comprising CDR1 , CDR2, and CDR3 from a VL sequence in Tables 1 -6 or 8 that is at least 90% identical to that VL sequence.
• an antibody according to the present disclosure comprises a heavy chain variable region comprising CDR1 , CDR2, and CDR3 from SEQ ID NO: 101 that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and/or comprises a light chain variable region comprising CDR1 , CDR2, and CDR3 from SEQ ID NO: 100 that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 100.
• the present disclosure provides an antibody comprising: (A) a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; 18, 19 and 20; 34, 35, and 36; 50, 51 , and 52; 66, 67, and 68 or 82, 83, and 84; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; 24, 25 and 26; 40, 41 and 42; 56, 57 and 58; 72, 73 and 74 or 88, 89 and 90; or (B) a VH and VL comprising the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 16; 30 and 32; 46 and 48; 62 and 64; 78 and 80 or 94 and 96, respectively
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10.
• HC heavy chain
• LC light chain
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 18, 19 and 20; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 24, 25 and 26.
• HC heavy chain
• LC-CDR3 light chain variable region
• LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 24, 25 and 26.
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 34, 35, and 36; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 40, 41 and 42.
• HC heavy chain
• LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 40, 41 and 42.
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 50, 51 , and 52; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58.
• HC heavy chain
• LC-CDR1 light chain variable region
• LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58.
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 66, 67, and 68; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 72, 73 and 74.
• HC heavy chain
• LC-CDR1 light chain variable region
• LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 72, 73 and 74.
• the antibody comprises a heavy chain (HC) variable region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 82, 83, and 84; and a light chain (LC) variable region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 88, 89 and 90.
• HC heavy chain
• LC-CDR1 light chain variable region
• LC-CDR1 , LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 88, 89 and 90.
• the antibody comprises a light chain consisting essentially of the amino acid sequence of SEQ ID NO: 100 and a heavy chain consisting essentially of the amino acid sequence of SEQ I D NO: 101 .
• an antibody of the present disclosure specifically binds to WT-1 peptide RMFPNAPYL (SEQ ID NO: 1 ) in conjunction with HLA/A2.
• the HLA-A2 is HLA-A0201 .
• the antibody exhibits between 50-1 00% (80%) higher affinity for activating human FcyRllla (158V variant) than normally glycosylated antibody, has 3- to 4-fold (3.5-fold) higher affinity for a FcyRllla 158F variant than normally glycosylated antibody, and has between 30 and 70% (50%) reduced affinity for inhibitory FcyRllb than normally glycosylated antibody.
• the present disclosure provides an isolated nucleic acid that encodes an antibody described herein, a vector comprising said nucleic acid, and a cell comprising said nucleic acid or said vector.
• the present disclosure provides a kit comprising an antibody described herein.
• the invention relates to a derivative or analog of an antibody of the present disclosure.
• a derivative can comprise any molecule or substance that imparts a desired property, such as increased half-life in a particular use.
• molecules that can be used to form a derivative include, but are not limited to, albumin (e.g., human serum albumin) and polyethylene glycol (PEG).
• albumin e.g., human serum albumin
• PEG polyethylene glycol
• Derivatives such as albumin-linked and PEGylated derivatives of antibodies can be prepared using techniques well known in the art.
• An analog may be a non-peptide analog of an antibody described herein. Non-peptide analogs are commonly used in the
• peptide mimetics or “peptidomimetics,” (Fauchere, J. Adv. Drug Res 1986;15:29; Veber and Freidinger TINS 1985;p. 392; Evans et al. J. Med. Chem 1987;30:1229).
• Peptide mimetics that are structurally similar to the antibodies of the present disclosure may be used to produce an equivalent therapeutic or prophylactic effect.
• a paradigm polypeptide i.e., a polypeptide that has a desired biochemical property or pharmacological activity
• Antibodies according to the present disclosure may be prepared by any of a number of conventional techniques. For example, they may be produced in recombinant expression systems, using any technique known in the art. See, for example,
• Monoclonal Antibodies, Hybridomas A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
• Certain of the techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an antibody of interest, and manipulating the nucleic acid through recombinant DNA technology.
• the nucleic acid may be fused to another nucleic acid of interest, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues.
• Any expression system known in the art can be used to make the recombinant antibodies of the present disclosure.
• host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired
• prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli.
• Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651 ) (Gluzman et al., Cell
• L cells L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the
• CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., EMBO J 1991 ; 10: 2821 .
• Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in the art, e.g., by Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985.
• the transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide recovered by conventional protein purification procedures.
• One such purification procedure includes the use of affinity chromatography, e.g., over a matrix having all or a portion of the antigen bound thereto.
• Polypeptides contemplated for use herein include substantially homogeneous
• the resulting antibody has an amino acid sequence as described above and no detectable fucose or galactose as part of the carbohydrate of the antibody.
• the fucose content and/or galactose content of the antibody can be reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to the wildtype antibody.
• a pharmaceutical composition of the present disclosure provides a pharmaceutical composition comprising an antibody described herein and a physiologically acceptable diluent, excipient, or carrier.
• a pharmaceutical composition of the present disclosure comprises a antibody described herein with one or more substances selected from the group consisting of a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having fewer than 10 amino acids), a protein, an amino acid, a carbohydrate, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient.
• Neutral buffered saline or saline mixed with serum albumin are examples of appropriate diluents.
• a liquid pharmaceutical composition may include, for example, one or more of the following: a sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils that may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents; antioxidants; chelating agents; buffers and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• an injectable pharmaceutical composition is preferably sterile.
• the composition may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.
• appropriate excipient solutions e.g., sucrose
• Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components that may be employed in pharmaceutical formulations are presented in Remington's Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed.
• compositions comprising the antibodies of the present disclosure are administered to a subject in a manner
• a pharmaceutical composition of the present disclosure comprising an antibody described herein may be formulated for delivery by any route that provides an effective dose of the antibody.
• Pharmaceutical compositions may be administered by any suitable technique, including but not limited to, parenterally, topically, or by inhalation. If injected, the pharmaceutical composition can be administered, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by bolus injection, or continuous infusion. Localized administration, e.g., at a tumor site, is contemplated, as are transdermal delivery and sustained release from implants.
• Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like.
• Other alternatives include eyedrops; oral preparations including tablets, capsules, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, patches, and ointments.
• the present disclosure provides use of an antibody described herein, e.g., in the preparation of a medicament, for the treatment of a WT1 positive disease.
• the present disclosure provides a method for treatment of a subject having a WT1 -positive disease, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment described herein.
• the WT1 -positive disease is a chronic leukemia or acute leukemia or a WT1 + cancer, for example, a WT1 -positive disease selected from the group consisting of chronic myelocytic leukemia, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma
• MM multiple myeloma
• ALL acute lymphoblastic leukemia
• AML acute myeloid/myelogenous leukemia
• MDS myelodysplastic syndrome
• a therapeutically effective amount of an antibody or pharmaceutical composition of the invention is an amount effective to inhibit growth of WT1 -positive cells, reduce tumor size/burden, prevent tumor cell metastasis/infiltration, and/or result in cell death, e.g., via apoptosis or necrosis.
• An antibody or pharmaceutical composition described herein need not effect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent.
• therapeutic agents may reduce the severity of a given disease state, but need not abolish every
• Dosages and the frequency of administration for use in the methods of the present disclosure may vary according to such factors as the route of administration, the particular antibodies employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject. Appropriate dosages can be determined by procedures known in the pertinent art, e.g., in clinical trials that may involve dose escalation studies.
• An antibody of the present disclosure may be administered, for example, once or more than once, e.g., at regular intervals over a period of time.
• the antibody or pharmaceutical composition is administered to a subject until the subject manifests a medically relevant degree of improvement over baseline for the chosen indicator or indicators.
• the amount of an antibody described herein present in a dose, or produced in situ by an encoding polynucleotide present in a dose ranges from about 10 iQ per kg to about 20 mg per kg of host.
• the use of the minimum dosage that is sufficient to provide effective therapy is usually preferred.
• Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented; assays will be familiar to those having ordinary skill in the art and some are described herein.
• the methods disclosed herein may include oral administration of an antibody described herein or delivery by injection of a liquid pharmaceutical composition.
• suitable dose sizes will vary with the size of the subject, but will typically range from about 1 ml to about 500 ml (comprising from about 0.01 iQ to about 1000 g per kg) for a 10kg to 60 kg subject.
• Optimal doses may generally be determined using experimental models and/or clinical trials. The optimal dose may depend upon the body mass, body area, weight, or blood volume of the subject. As described herein, the appropriate dose may also depend upon the patient's condition, that is, stage of the disease, general health status, age, gender, weight, and other factors familiar to a person skilled in the medical art.
• the subject is a human or non-human animal.
• a subject in need of the treatments described herein may exhibit symptoms or sequelae of a disease, disorder, or condition described herein or may be at risk of developing the disease, disorder, or condition.
• Non-human animals that may be treated include mammals, for example, non-human primates (e.g., monkey, chimpanzee, gorilla), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic farm and zoo animals.
• non-human primates e.g., monkey, chimpanzee, gorilla
• rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
• lagomorphs e.g., pig, miniature pig
• swine
• N-Glycan from ESK1 or ESKM antibodies was cleaved from antibody by PNGase F, and measured by HPAEC-PAD using PA200 column. Binding of ESK1 /ESKM antibodies to mouse FcyR4 and mouse FcyRllb were measured by ELISA. Briefly, 2 ⁇ g/mL recombinant mouse FcyR4 or FcyRllb were coated onto ELISA plate. Various concentrations of ESK1 or ESKM antibodies were added to the wells for 1 hour at room temperature, then detected by secondary antibody (HRP conjugated anti- human IgG Fab'2 fragment).
• Binding of ESK1 /ESKM to human FcyRs was measured by Flow Cytometry (Guava easyCyte HT, Millipore) against CHO cells expressing appropriate human FcyR. Binding of ESK1 /ESKM to human FcyRI, FcyRlla, FcyRllla- 158V, FcyRllla-158F and human FcRn were measured directly using ESK1 or ESKM antibody, followed by the 2 nd antibody (FITC conjugated Fab'2 fragment anti-human IgG Fab'2).
• dimers were formed first by mixing ESK1 or ESKM to a PE- conjugated Fab'2 fragment anti-human Fab'2 at 2:1 ratio at RT for 2 hour. Binding of dimeric complex of ESK1 or ESKM to human FcyRllb were measured directly by Flow Cytometry using the immunocomplex.
• ESK1 was expressed in Chinese Hamster Ovary cells using the GlymaxX® technology (ProBioGen, Berlin, Germany) to reduce the fucose content of the antibody.
• ESKM antibody was purified from two separate pools of cells, and fucose reduction confirmed by mass spectrometry to be 70% reduced or 100% reduced. Both the ESKM having 70% reduction in fucosylation and the ESKM having 100% reduction in fucosylation (completely a-fucosylated) batches were compared to ESK1 as a wildtype lgG1 and to ESK1 containing D265A/P329A mutations in the Fc domain (ESK1 DAPA) that eliminated binding to human FcgRllla in ADCC killing assays.
• T2 cells were pulsed with 25 mg/ml RMFPNAPYL (SEQ ID NO:1 ) peptide overnight. The next day, 15,000 pulsed cells were added to serially diluted ESK-1 antibodies. Then 90,000 Jurkat cells transduced to express CD16A and an NFAT- luciferase reporter were added. Plates were gently mixed and spun at 200 x g for 4 minutes and then incubated at 37 °C for 4 hours. At the end of the incubation, the plates were brought to room temp for -15 minutes, 60 ⁇ of BrightliteTM (Perkin Elmer) was then added to each well. Plates were shaken for 3 minutes and analyzed on the EnVision Multilabel Reader (PerkinElmer).
• ADCC Antibody-dependent cellular cytotoxicity
• PBMCs Peripheral blood mononuclear cells from healthy donors were obtained by Ficoll density centrifugation.
• Target cells used for ADCC were T2 cells pulsed with or without WT1 or RHAMM-3 peptides, and cancer cell lines without peptide pulsing.
• ESK1 , ESKM or isotype control human lgG1 at various concentrations were incubated with target cells and fresh PBMCs at different effector: target (E:T) ratio. Cytotoxicity was measured by standard 4 hour 51 Cr-release assay.
• Luciferase-expressing JMN cells (3 x 10 5 ) were injected into the intraperitoneal cavity of CB17 SCID mice. On day 4, tumor engraftment was confirmed by luciferase imaging, signal was quantified with Living Image® software (Xenogen), and mice were sorted into groups with similar average signal from the supine position. Mice were injected intraperitoneal ⁇ with 50 g ESK1 , ESKM or human isotype lgG1 antibody twice weekly beginning on day 4. [000106] For AML leukemia studies, luciferase-expressing BV173 (Ph+ ALL) or SET2 (AML) cells (3 x 10 6 ) were injected intravenously via tail vein into NSG mice. Animals were sorted, and, where indicated, treated with intraperitoneal injections of 100 ⁇ g ESKM twice weekly beginning on day 6.
• ESKM or isotype control antibody 100 ⁇ g/animal was administered via retro-orbital injection on days 2, 5, 9, 12, 14 and 23, and leukemia growth was followed by bioluminescent imaging.
• animals were sacrificed and bone marrow cells were harvested and pooled: after dissection and homogenization, cells were centrifuged, subjected to Ficoll density centrifugation, and counted after red blood cell lysis (acetic acid).
• Antibody was labeled with 125 l (PerkinElmer) using the chloramine-T method. 100 ⁇ g antibody was reacted with 1 mCi 125 l and 20 ⁇ g chloramine-T, quenched with 200 ⁇ g Na metabisulfite, then separated from free 125 l using a 10DG column equilibrated with 2% bovine serum albumin in PBS. Specific activities of products were in the range of 4-8 mCi/mg. Radiolabeled mAb was injected into mice retro-orbitally, and blood and/or organs were collected at various time points, weighed and measured on a gamma counter.
• C57BL6/J or HLA-A2.1 + transgenic mice were sacrificed, and cells were harvested from spleen, thymus and bone-marrow. After red blood cell lysis, cells (10 6 per tube, in duplicate) were incubated with 125 l-labeled ESK1 (1 pg/ml) for 45 minutes on ice, then washed extensively with 1 % bovine serum albumin in PBS on ice. To determine specific binding, a set of cells was assayed after pre-incubation in the presence of 50-fold excess unlabeled ESK1 for 20 minutes on ice. Bound radioactivity was measured by a gamma counter, specific binding was determined, and the number of bound antibodies per cell was calculated from specific activity.
• mice treated as above were sacrificed on day 6 for histopathologic examination of major organs and possible WT1 positive target organs (spleen, bone and bone marrow, liver, thymus, kidney) as well as heart, lung, and ileum. Mice were sacrificed and whole organs were collected, fixed (4% paraformaldehyde), decalcified in EDTA where necessary (femurs only), embedded in paraffin, sectioned and stained with H&E.
• Antibodies and flow cytometry analysis [0001 12] For cell surface staining, cells were incubated with appropriate mAbs for SO- SO minutes on ice, washed, and incubated with secondary antibody reagents when necessary. Flow cytometry data were collected on a FACS Calibur or LSRFortessa (Becton Dickinson) and analyzed with FlowJo software. APC-labeled ESK1 and hlgG1 isotype antibodies were generated with Lightning-Link® kit (Innova Biosciences).
• mouse bone marrow cells were stained with the following antibodies: (Lineage; CD3, CD4, CD8, Gr1 , B220, CD19, TER1 19, all conjugated with PE-Cy5), Sea- Pacific Blue, CD34-FITC, SLAM-APC, CD48-PE and c- KIT-AlexaFluor 780.
• the stained cells were analyzed for flow cytometry on the BD LSRII instrument.
• ESKM antibody has enhanced binding affinity for FcyRllla and reduced affinity for FcyRllb
• ESKM mAb was produced in MAGE 1 .5 CHO cells, with the homogeneous oligosaccharide structure (Fig 1 A) and no detectable fucose or galactose.
• ESKM had 80% higher affinity for activating human FcyRllla (158V variant), 3.5-fold higher affinity for the FcyRllla 158F variant, and 50% reduced affinity for inhibitory FcyRllb (Table 9)
• ESKM showed enhanced reverse signaling through FcyRIIIA (CD16A) compared to wildtype ESK1 , indicating improved binding interaction.
• An approximate 5- fold decrease in EC50 was observed with ESKM relative to wildtype ESK1 (EC50 values: 0.17 for ESKM; 0.88 for ESK1 wildtype; >10000 for ESK1 DAPA) (Fig. 1 H).
• ESKM effectively mediated ADCC against BA-25, an acute lymphoblastic leukemia (ALL) cell line expressing approximately 1000-2000 RMF/A2 targets per cell; both antibodies were similarly effective at ADCC at concentrations above 1 pg/mL, but ESKM was more potent at concentrations down to 100 ng/ml of mAb (Fig 2B).
• ALL acute lymphoblastic leukemia
• ESKM was more potent at concentrations down to 100 ng/ml of mAb
• Fig 2B Against AML-14 and SET2 acute myeloid leukemia (AML) cell lines, which both bind -5000 mAb per cell, ESKM mediated higher specific cell lysis than ESK1 at the highest antibody concentrations, and showed cytolytic efficacy down to doses as low as 100 ng/ml (Fig. 1 C-1 D).
• Murine FcyRIV Human Fc can engage murine FcyRIV (Pietzsch J et al. Proceedings of the National Academy of Sciences of the United States of America 2012 ; 109 (39) : 15859- 64), therefore murine NK cells should serve as potent effectors in vivo; as ESKM has enhanced binding to murine FcyRIV, it was expected to be more efficacious than the native mAb in this model.
• Mice were engrafted with luciferase+ JMN mesothelioma cells in the intraperitoneal cavity (simulating this serosal cavity cancer). To determine the relative abundance of effector cell populations in the intraperitoneal cavity, extracted cells with common murine immunophenotyping markers (Lai L et al.
• ESKM was also investigated in the luciferase+ SET2 mouse model of AML.
• the SET2 cell line grew much faster than BV1 73 in the NSG mouse model and disseminated throughout the mouse bone marrow.
• ESKM was able to significantly reduce tumor growth (Fig. 3D).
• a fresh human pre-B-cell ALL derived from a CNS-relapse was engrafted into NSG mice.
• ESKM significantly reduced initial leukemia burden and slowed leukemia outgrowth (Fig. 3E and 3F). Leukemia relapsed after treatment was stopped (Fig.
• Fig. 3F allowing for leukemia cells to be collected from the bone marrow and transplanted to new animals to assess outgrowth from remaining progenitors
• Fig. 3G Total bone marrow signal in ESKM-treated mice was lower at time of transplant (Fig. 3H), but equal numbers of ESKM-treated and isotype-treated bone marrow cells were engrafted into recipient animals.
• Subcutaneous leukemia tumors from isotype-treated leukemia cells grew 20 times faster than from ESKM-treated cells (Fig. 31).
• the C57BL6/J mouse model cannot recapitulate possible on-target binding to normal tissues that could alter antibody pharmacokinetics and biodistribution. Therefore, a transgenic mouse model based on the C57BL6/J background that expresses human HLA-A201 driven by a lymphoid promoter was used.
• the 9-mer RMF sequence is identical in human and mouse, and therefore this transgenic model could recapitulate antigen presentation of the RMF/HLA-A0201 epitope in various healthy organs.
• WT1 is reported to be expressed in hematopoetic stem cells (HSC) (Ariyaratana S and Loeb DM. Expert reviews in molecular medicine 2007;9(14):1 -17), so the C57 BL6/J transgenic mouse model with human HLA-A0201 driven by a lymphoid promoter provides an opportunity to assess possible toxicity against progenitor cells in the hematopoetic compartment that, given the high potency of ESKM, might occur even at low epitope density.
• HSC hematopoetic stem cells
• Table 1 1 spleen thymus liver kidney lung Gl heart bone
• hematopoiesis with adequate maturation of the myeloid and erythroid lineages.
• the megakaryocytes were adequate in number with normal morphology.
• Thymus sections showed a well-defined cortex and medulla with few Hassall's corpuscles, which is normal for rodent histology.
• the kidney sections showed no pathologic findings such as glomerulosclerosis, congestion, or inflammation.
• Liver sections showed normal lobular architecture without congestion or inflammation. All spleen sections showed a normal distribution of red and white pulp. Occasional scattered megakaryocytes were seen in the red pulp consistent with extramedullar hematopoiesis (Cesta MF. Toxicologic pathology 2006 ;34(5):455-65).
• TCRm antibodies allow use of the mAb to target cell- surface fragments of intracellular proteins, provided that they are processed and presented on MHC class I molecules.
• ESK1 was the first TCRm antibody reported against a peptide derived from WT1 , an important oncogene expressed in a wide variety of cancers, but not normal adult tissues.
• WT1 appears to be expressed in leukemic stem cells (Ariyaratana S and Loeb DM. Expert reviews in molecular medicine 2007;9(14):1 -17), raising the possibility that the mAb could ultimately eliminate clonogenic leukemia cells in patients.
• Other therapeutic TCRm mouse antibodies, human ScFv and Fab fragments have been previously described (Epel M et al. European journal of immunology 2008;38(6):1706-20; Wittman VP et al. J Immunol 2006;177(6):4187-95; Klechevsky et al. Cancer research 2008;68(15):6360-7; Verma B, et al. J Immunol 2010;184(4):2156-65; Sergeeva et al. Blood 201 1 ;1 17(16):4262-72).
• ESK1 is the first and only fully human therapeutic TCRm mAb reported.
• ESKM mAb had a homogeneous glycosylation pattern lacking N-linked fucose and with terminal hexose (mannose and/or glucose) structure.
• This engineering strategy modulates mAb binding to Fey receptors in two ways: a higher affinity for activating human FcYRIIIa (and murine FcyRIV) increases ADCC activity; while diminished affinity for both human and murine FcyRllb should reduce inhibitory receptor activation.
• ESKM was both more potent and effective in vitro even at very low epitope density.
• ESKM was also more effective than ESK1 in vivo, and was able to treat peritoneal mesothelioma in SCID mice, modeling the clinical situation. Three of 5 animals displayed absolute reduction in tumor burden over the two-week treatment course, whereas none of the ESK1 -treated mice achieved more than a slowing of initial tumor growth. After termination of therapy, ESKM-treated mice survived longer than ESK1 treatment groups, with 1 of 5 animals surviving without disease. Further, ESKM significantly slowed leukemia growth of disseminated SET2, an AML cell line with much more aggressive in vivo leukemia growth kinetics than BV173, and a fresh patient- derived pre-B-cell ALL in xenograft models.
• ESKM may target a progenitor population of leukemia cells, which is consistent with the hypothesis that WT1 expression in HSCs could allow ablation of this population.
• cells collected from the bone marrow were not phenotyped and sorted, so the exact cell population targeted was not determined.
• ESKM therapy was not effective against peritoneal mesothelioma in NSG or NOG mice, which lack NK-cells, though naked ESK1 did previously show potent activity against a disseminated leukemia model in these mice. This discrepancy could be due both to the tumor model— leukemia cells could have different sensitivity to effector- mediated cytotoxicity— and to the availability of effector cells in the NSG/NOG model.
• ESK-bound target cells is likely more optimal in the circulation and hematopoetic compartments, where the leukemia grew, than in the peritoneal cavity; further, assays indicated that the intraperitoneal cavity of NOG mice contained predominately macrophages, while neutrophils were present in the blood and spleen.
• Altering Fc glycosylation could potentially change pharmacokinetic properties of the mAb through a number of mechanisms, including: altered FcRn binding and antibody recycling, modified binding to circulating effector cells, and differential engagement with clearance mechanisms, such as mannose receptors. Similar afucosylated, Fc-modified antibodies with improved ADCC have been investigated in pharmacokinetic studies in vivo (Gasdaska JR et al. Molecular immunology 2012;50(3):134-41 , Junttila TT et al. Cancer research 2010;70(1 1 ):4481 -9). ESKM had nearly identical biodistribution to ESK1 , but a shortened blood half-life.
• ESK mAbs target a human HLA-specific epitope
• the human HLA- A0201 + transgenic mouse strain was utilized for toxicology studies.
• WT1 is reportedly expressed in HSCs, yet a therapeutic dose of ESKM that cleared leukemia in the models had no effect on LSK cells or early HSCs.
• organ histology was normal.
• ESKM did not affect the architecture or cell coverage in the bone marrow, thymus or spleen, where WT1 + HSCs could be expected, and where HLA-A0201 expression is highest because the transgene is driven by a lymphoid promoter.
• ESKM has moderately decreased half-life yet increased potency and broader applicability.
• the potential enhanced efficacy against tumors expressing fewer RMF/A2 sites could expand the number of patients and cancer types eligible for this therapy as well as increase efficacy.
• the MAGE 1 .5 CHO engineering technology generates mAbs that effectively engage FcYRIIIa (CD16), regardless of amino acid 158 polymorphism.
• Carriers of CD16-158F are less responsive than CD16- 158V/V individuals to human lgG1 therapeutics such as rituximab and trastuzumab (Cartron G et al. Blood 2002 ;99(3):754-8, Musolino A et al.
• ESKM is a potent therapeutic mAb against a widely expressed oncogenic target with a restricted normal cell expression profile, and has shown efficacy against multiple human tumor models in mice.

Applications Claiming Priority (2)

Publications (1)

Family

ID=52021419

Family Applications (1)

Country Status (4)

Families Citing this family (6)

Family Cites Families (3)

• 2014
  • 2014-11-07 AU AU2014346648A patent/AU2014346648A1/en not_active Abandoned
  • 2014-11-07 CA CA2934033A patent/CA2934033A1/en not_active Abandoned
  • 2014-11-07 WO PCT/US2014/064657 patent/WO2015070078A1/en active Application Filing
  • 2014-11-07 EP EP14810051.4A patent/EP3066131A1/de not_active Withdrawn

Also Published As

Similar Documents

Legal Events