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WO2019084067A1 - Anticorps anti-cd117 et leurs méthodes d'utilisation - Google Patents

Anticorps anti-cd117 et leurs méthodes d'utilisation

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Publication number
WO2019084067A1
WO2019084067A1 PCT/US2018/057185 US2018057185W WO2019084067A1 WO 2019084067 A1 WO2019084067 A1 WO 2019084067A1 US 2018057185 W US2018057185 W US 2018057185W WO 2019084067 A1 WO2019084067 A1 WO 2019084067A1
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WO
WIPO (PCT)
Prior art keywords
seq
amino acid
set forth
acid sequence
antibody
Prior art date
Application number
PCT/US2018/057185
Other languages
English (en)
Inventor
Bradley R. PEARSE
Anthony Boitano
Rahul Palchaudhuri
Sean MCDONOUGH
Rajiv PANWAR
Jonathan Philip BELK
Matthew Duncan SMITH
Original Assignee
Magenta Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magenta Therapeutics, Inc. filed Critical Magenta Therapeutics, Inc.
Publication of WO2019084067A1 publication Critical patent/WO2019084067A1/fr

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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • 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
    • 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
    • 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
    • C07K2317/77—Internalization into the cell
    • 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
    • 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

Definitions

  • the invention relates to anti-CD1 17 antibodies and antigen binding fragments thereof as well as the treatment of patients suffering from various pathologies, such as blood diseases, metabolic disorders, cancers, and autoimmune diseases, among others, by administration of an antibody, antigen-binding fragment thereof, capable of binding an antigen expressed by a hematopoietic cell, such as a hematopoietic stem cell.
  • hematopoietic stem cells have significant therapeutic potential, a limitation that has hindered their use in the clinic has been the difficulty associated with ensuring engraftment of hematopoietic stem cell transplants in a host.
  • compositions and methods for promoting the engraftment of exogenous hematopoietic stem cell grafts such that the multi-potency and hematopoietic functionality of these cells is preserved following transplantation.
  • compositions that target specific endogenous stem cells that can be used as conditioning agents to promote the engraftment of exogenous hematopoietic stem cell grafts such that the multi-potency and hematopoietic functionality of these cells is preserved in the patient following transplantation.
  • CD1 17 (also referred to as c-kit or Stem Cell Factor Receptor (SCRF)) is a single transmembrane, receptor tyrosine kinase that binds the ligand Stem Cell Factor (SCF) . SCF induces homodimerization of cKIT which activates its tyrosine kinase activity and signals through both the PI3-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998 152(5):1259).
  • SCF Stem Cell Factor Receptor
  • CD1 17 was initially discovered as an oncogene and has been studied in the field of oncology (see, for example, Stankov et al. (2014) Curr Pharm Des. 20(17):2849-80).
  • An antibody drug conjugate (KTN0158) directed to CD1 17 is currently under investigation for the treatment of refractory gastrointestinal stromal tumors (GIST) (e.g., "KTN0158, a humanized anti-KIT monoclonal antibody, demonstrates biologic activity against both normal and malignant canine mast cells" London et al. (2016) Clin Cancer Res DOI: 10.1 158/1078-0432.CCR-16-2152).
  • GIST refractory gastrointestinal stromal tumors
  • CD1 17 is highly expressed on hematopoietic stem cells (HSCs). This expression pattern makes CD1 17 a potential target for conditioning across a broad range of diseases. There remains, however, a need for anti-CD1 17 based therapy that is effective for conditioning a patient for transplantation, such as a bone marrow transplantation.
  • HSCs hematopoietic stem cells
  • Described herein are antibodies, and antigen binding portions thereof, that specifically bind human CD1 17 (also known as c-kit), as well as compositions and methods of using said antibodies.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9.
  • an anti-CD1 17 antibody or antigen-binding portion thereof, comprises a light chain variable region comprising a CDR1 domain as set forth in SEQ ID NO: 14, a CDR2 domain as set forth in SEQ ID NO: 15, and a CDR3 domain as set forth in SEQ ID NO: 16, and comprises a heavy chain variable region comprising a CDR1 domain as set forth in SEQ ID NO: 1 1 , a CDR2 domain as set forth in SEQ ID NO: 12, and a CDR3 domain as set forth in SEQ ID NO: 13.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 31 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:32, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 33; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 34, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:35, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 36.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 21 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:22, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 23; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 24, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:25, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 26.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 41 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:42, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 43; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 44, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:45, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 46.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 51 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:52, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 53; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 54, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:55, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 56.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 61 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:62, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 63; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 64, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:65, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 66.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 71 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:72, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 73; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 74, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:75, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 76.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 81 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:82, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 83; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 84, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:85, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 86.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 1 1 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:12, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 13; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 14, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:15, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 16.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 91 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:92, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 93; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 94, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:95, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 96.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 101 , a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:102, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 103; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 104, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:105, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 106.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 127, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:128, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 129; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 130, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:131 , and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 132.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 133, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:134, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 135; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 136, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:137, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 138.
  • an anti-CD1 17 antibody comprises a heavy chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 139, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:140, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 141 ; and comprising a light chain variable region comprising a CDR1 domain comprising the amino acid sequence as set forth in SEQ ID NO: 142, a CDR2 domain comprising the amino acid sequence as set forth in SEQ ID NO:143, and a CDR3 domain comprising the amino acid sequence as set forth in SEQ ID NO: 144.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 29, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 30.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 19, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 20.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 40.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 49, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 50.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 59, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 60.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 69, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 70.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 80.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 10.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 89, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 90.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 99, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 100.
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises an Fc region comprising amino acid substitution D265C (numbering according to EU index).
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises an Fc region comprising amino acid substitutions D265C, L234A, and L2345A
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises an Fc region comprising amino acid substitutions D265C and H435A (numbering according to EU index). In some embodiments, the anti-CD1 17 antibody or antigen-binding fragment thereof comprises an Fc region comprising amino acid substitutions D265C, L234A, L2345A, and H435A (numbering according to EU index).
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises a (i) light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 121 , and (ii) a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 122.
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises (i) a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 121 , and (ii) a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 123.
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises (i) a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 121 , and (ii) a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 124.
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises (i) a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 121 , and (ii) a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 125.
  • the anti-CD1 17 antibody or antigen-binding fragment thereof comprises (i) a light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 121 , and (ii) a heavy chain constant region comprising the amino acid sequence as set forth in SEQ ID NO: 126.
  • Antibody 54 (Ab54), Antibody 55 (Ab55), Antibody 56 (Ab56), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61 ),
  • Antibody 66 (Ab66), Antibody 67 (Ab67), Antibody 68 (Ab68), or Antibody 69 (Ab69) may be used in compositions and methods for the treatment of various disorders of the hematopoietic system, metabolic disorders, cancers, and autoimmune diseases, among others.
  • the invention additionally features methods for conditioning a patient, such as a human patient, prior to receiving hematopoietic stem cell transplant therapy so as to promote the engraftment of hematopoietic stem cell grafts.
  • the patient may be one that is suffering from one or more blood disorders, such as a hemoglobinopathy or other hematopoietic pathology, and is thus in need of hematopoietic stem cell transplantation.
  • hematopoietic stem cells are capable of differentiating into a multitude of cell types in the hematopoietic lineage, and can be administered to a patient in order to populate or re-populate a cell type that is deficient in the patient.
  • the invention features methods of treating a patient with antibodies capable of binding proteins expressed by hematopoietic cells, such as CD1 17 (including, for example, GNNK+ CD1 17), so as to (i) directly treat a disease such as a blood disorder, metabolic disease, cancer, or autoimmune disease, among others described herein, by selectively depleting a population of cells that express CD1 17, such as an aberrant blood cell, cancer cell, or autoimmune cell, and/or (ii) deplete a population of endogenous hematopoietic stem cells within the patient.
  • a disease such as a blood disorder, metabolic disease, cancer, or autoimmune disease, among others described herein
  • CD1 17 including, for example, GNNK+ CD1 17
  • the former activity enables the direct treatment of a wide range of disorders associated with a cell of the hematopoietic lineage, as CD1 17 may be expressed by a cancerous cell, such as a leukemic cell, an autoimmune lymphocyte, such as a T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen, among other cell types.
  • a cancerous cell such as a leukemic cell
  • an autoimmune lymphocyte such as a T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen, among other cell types.
  • the latter activity the selective depletion of hematopoietic stem cells, in turn creates a vacancy that can subsequently be filled by
  • the invention thus provides methods of treating a variety of hematopoietic conditions, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott- Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
  • hematopoietic conditions such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott- Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among
  • the invention provides a method of depleting a population of CD1 17+ cells in a human patient by administering an effective amount of an antibody or antigen-binding fragment thereof capable of binding CD1 17.
  • the invention provides a method of depleting a population of CD1 17+ cells in a human patient in need of a hematopoietic stem cell transplant by administering, prior to the patient receiving a transplant including hematopoietic stem cells, an effective amount of an antibody or antigen-binding fragment thereof capable of binding CD1 17.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including administering to a human patient a transplant including hematopoietic stem cells, wherein the patient has been previously administered an antibody or antigen-binding fragment thereof capable of binding CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including: administering to a human patient an antibody or antigen-binding fragment thereof capable of binding CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient, and subsequently administering to the patient a transplant including hematopoietic stem cells.
  • the CD1 17 is GNNK+ CD1 17.
  • the invention provides a method of depleting a population of CD1 17+ cells in a human patient in need of a hematopoietic stem cell transplant by administering, prior to the patient receiving a transplant including hematopoietic stem cells, an effective amount of an antibody or fragment thereof capable of binding CD1 17.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including administering to a human patient a transplant including hematopoietic stem cells, wherein the patient has been previously administered an antibody or fragment thereof capable of binding CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including: administering to a human patient an antibody or fragment thereof capable of binding CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient, and subsequently administering to the patient a transplant including hematopoietic stem cells.
  • the antibody or fragment thereof that binds CD1 17 is covalently bound to an Fc domain, such as a dimeric Fc domain isolated from a human antibody (for example, isolated from an lgG1 , lgG2, lgG3, or lgG4 isotype human antibody).
  • the Fc domain is a monomeric Fc domain containing a single polypeptide strand.
  • the N-terminus of the antibody or fragment thereof is bound to the Fc domain.
  • the C-terminus of the antibody or fragment thereof is bound to the Fc domain.
  • the Fc domain is a human lgG1 isotype Fc domain. In some embodiments, the Fc domain is a human lgG2 isotype Fc domain. In some embodiments, the Fc domain is a human lgG3 isotype Fc domain. In some embodiments, the Fc domain is a human lgG4 isotype Fc domain.
  • the invention features a method of depleting a population of CD1 17+ cells in a human patient by administering an effective amount of an antibody, or antigen-binding fragment thereof capable of binding GNNK+ CD1 17.
  • the invention features a method of depleting a population of CD1 17+ cells in a human patient in need of a hematopoietic stem cell transplant by administering, prior to the patient receiving a transplant containing hematopoietic stem cells, an effective amount of an antibody, or antigen-binding fragment thereof capable of binding GNNK+ CD1 17.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including administering to a human patient a transplant containing hematopoietic stem cells, wherein the patient has been previously administered an antibody, or antigen-binding fragment thereof capable of binding GNNK+ CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient.
  • the invention features a method, for example, of treating a human patient in need of a hematopoietic stem cell transplant, including: administering to a human patient an antibody, or antigen-binding fragment thereof capable of binding GNNK+ CD1 17 in an amount sufficient to deplete a population of CD1 17+ cells in the patient, and subsequently administering to the patient a transplant including hematopoietic stem cells.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab') 2 molecule, and a tandem di-scFv.
  • the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.
  • the antibody, or antigen-binding fragment thereof is internalized by a hematopoietic cell, such as a hematopoietic stem cell, cancer cell, or autoimmune cell following administration to the patient.
  • a hematopoietic cell such as a hematopoietic stem cell, cancer cell, or autoimmune cell following administration to the patient.
  • the antibody, or antigen-binding fragment thereof may be internalized by hematopoietic stem cells, cancer cells, or autoimmune cells by receptor-mediated endocytosis (e.g., upon binding to cell-surface CD1 17, such as GNNK+ CD1 17).
  • the antibody, or antigen-binding fragment thereof is capable of promoting necrosis of a hematopoietic cell, such as a
  • the antibody or antigen-binding fragment thereof may promote the death of an endogenous hematopoietic stem cell prior to transplantation therapy, an endogenous cancer cell, or an endogenous autoimmune cell, among others, by recruiting one or more complement proteins, natural killer (NK) cells, macrophages, neutrophils, and/or eosinophils to the cell, such as a hematopoietic stem cell upon administration to the patient.
  • NK natural killer
  • the transplant containing hematopoietic stem cells is administered to the patient after the concentration of the antibody or antigen-binding fragment thereof has substantially cleared from the blood of the patient.
  • the hematopoietic stem cells or progeny thereof maintain hematopoietic stem cell functional potential after two or more days (for example, from about 2 to about 5 days, from about 2 to about 7 days, from about 2 to about 20 days, from about 2 to about 30 days, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more) following transplantation of the hematopoietic stem cells into the patient.
  • days for example, from about 2 to about 5 days, from about 2 to about 7 days, from about 2 to about 20 days, from about 2 to about 30 days, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13
  • the hematopoietic stem cells or progeny thereof are capable of localizing to hematopoietic tissue, such as the bone marrow, and/or reestablishing hematopoiesis following transplantation of the hematopoietic stem cells into the patient.
  • the hematopoietic stem cells upon transplantation into the patient, give rise to recovery of a population of cells selected from the group consisting of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen- presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B- lymphocytes.
  • a population of cells selected from the group consisting of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen- presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B- lymphocytes.
  • the method is used to treat one or more disorders, such as by depleting a population of hematopoietic stem cells in a patient prior to hematopoietic stem cell transplant therapy so as to provide a niche to which the transplanted hematopoietic stem cells may home.
  • the hematopoietic stem cells may establish productive hematopoiesis, so as to replenish a deficient cell type in the patient or a cell type that is being actively killed or has been killed, for instance, by chemotherapeutic methods.
  • the patient may be one that is suffering from a stem cell disorder.
  • the patient is suffering from a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • a hemoglobinopathy disorder such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • the patient may be suffering from an immunodeficiency disorder, such as a congenital
  • immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human
  • the patient is suffering from a metabolic disorder, such as glycogen storage diseases,
  • the patient is suffering from a disorder selected from the group consisting of adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis.
  • the patient is suffering from an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, ant Type 1 diabetes.
  • the patient is suffering from cancer or myeloproliferative disease, such as a hematological cancer.
  • the patient is suffering from acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the patient is suffering from a myelodysplastic disease, such as myelodysplastic syndrome.
  • the method is used to directly treat a cancer, such as a cancer characterized by CD1 17+ cells (e.g., a leukemia characterized by CD1 17+ cells), by administration of an antibody, or antigen-binding fragment thereof, that depletes a population of CD1 17+ cancer cells in the patient and/or by administration of an antibody, or antigen-binding fragment thereof, so as to deplete a population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation.
  • the transplantation may in turn re-constitute, for example, a population of cells depleted during the process of eradicating cancer cells.
  • the cancer may be a hematological cancer, such as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the method is used to treat an autoimmune disease, such as by administration of an antibody, or antigen-binding fragment thereof, so as to deplete a population of CD1 17+ autoimmune cells and/or by administration of an antibody, or antigen-binding fragment thereof, so as to deplete a population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation.
  • an autoimmune disease such as by administration of an antibody, or antigen-binding fragment thereof, so as to deplete a population of CD1 17+ autoimmune cells and/or by administration of an antibody, or antigen-binding fragment thereof, so as to deplete a population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation.
  • the transplantation may in turn re-constitute, for example, a population of cells depleted during the process of eradicating autoimmune cells.
  • the autoimmune disease may be, for example, scleroderma, multiple sclerosis (MS), human systemic lupus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes), acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitisis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliac
  • the invention features a method of treating a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • a hemoglobinopathy disorder such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • an immunodeficiency disorder such as a congenital immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human immunodeficiency virus or acquired immune deficiency syndrome).
  • the invention features a method of treating a metabolic disorder, such as glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
  • a metabolic disorder such as glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
  • the invention features a method of treating a disorder selected from the group consisting of adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis
  • the invention features a method of treating an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, ant Type 1 diabetes.
  • the invention features a method of treating a cancer or myeloproliferative disease, such as a hematological cancer.
  • the invention features a method of treating acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the patient is suffering from a myelodysplastic disease, such as myelodysplastic syndrome.
  • the method may include the steps of administering an antibody, or antigen-binding fragment thereof, that binds CD1 17 (e.g., GNNK+ CD1 17) and/or a
  • hematopoietic stem cell transplant according to the method of any of the above-described aspects and embodiments of the invention.
  • the invention provides a method of treating cancer directly, such as a cancer characterized by CD1 17+ cells (e.g., a leukemia characterized by CD1 17+ cells).
  • a cancer characterized by CD1 17+ cells e.g., a leukemia characterized by CD1 17+ cells.
  • the method includes
  • the cancer may be a hematological cancer, such as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the invention provides a method of treating an autoimmune disease, such as multiple sclerosis (MS), human systemic lupus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), treating psoriasis, Type 1 diabetes mellitus (Type 1 diabetes) acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitisis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatrical pebstructive fibrosis, atopic
  • the method includes administering an antibody, or antigen-binding fragment thereof, that binds CD1 17 (e.g., GNNK+ CD1 17).
  • the antibody or antigen-binding fragment thereof is internalized by a CD1 17+ cell.
  • the antibody or antigen-binding fragment thereof binds CD1 17 with a K d of less than 1 ⁇ , less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM.
  • the K d is from about 0.1 pM to about 1 ⁇ .
  • the antibody or antigen-binding fragment thereof binds CD1 17 with a k on of from about 9 x 10 "2 M “1 s “1 to about 1 x 10 2 M “1 s “1 .
  • the antibody or antigen-binding fragment thereof competitively inhibits the binding of CD1 17 to a second antibody or antigen binding fragment thereof, or binds the same epitope as a second antibody, wherein the second antibody or antigen- binding fragment thereof has the following complementarity determining regions (CDRs):
  • the antibody or antigen-binding fragment thereof competitively inhibits the binding of CD1 17 to a second antibody or antigen binding fragment thereof, or binds the same epitope as a second antibody, wherein the second antibody or antigen- binding fragment thereof has the following complementarity determining regions (CDRs) :
  • compositions and methods disclosed herein include anti-CD1 17 antibody Ab67.
  • compositions and methods disclosed herein include anti- CD1 1 7 antibody comprising a variable heavy chain amino acid sequence as set forth in SEQ ID NO: 9, and a light chain variable region comprising a light chain amino acid sequence as set forth in SEQ ID NO: 10.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab') 2 molecule, and a tandem di- scFV.
  • scFv single-chain Fv molecule
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab') 2 molecule, and a tandem di- scFv.
  • the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.
  • the invention features an antibody or fragment thereof that binds CD1 17 (e.g., GNNK+ CD1 17) covalently bound to an Fc domain, such as a dimeric Fc domain isolated from a human antibody (for example, isolated from an lgG1 , lgG2, lgG3, or lgG4 isotype human antibody).
  • an Fc domain such as a dimeric Fc domain isolated from a human antibody (for example, isolated from an lgG1 , lgG2, lgG3, or lgG4 isotype human antibody).
  • the Fc domain is a monomeric Fc domain containing a single polypeptide strand.
  • the N-terminus of the antibody or fragment thereof is bound to the Fc domain.
  • the C-terminus of the antibody or fragment thereof is bound to the Fc domain.
  • the Fc domain is a human lgG1 isotype Fc domain. In some embodiments of this aspect, the Fc domain is a human lgG2 isotype Fc domain. In some embodiments of this aspect, the Fc domain is a human lgG3 isotype Fc domain. In some embodiments of this aspect, the Fc domain is a human lgG4 isotype Fc domain.
  • the foregoing methods and compositions include an anti-CD1 17 antibody or antigen-binding fragment thereof comprising the CDRs set forth in the heavy and light chain amino acid sequences set forth in Table 10 for any one of Ab54, Ab55, Ab56, Ab57, Ab58, Ab61 , Ab66, Ab67, Ab68, or Ab69.
  • the foregoing methods and compositions include an anti-CD1 17 antibody or antigen-binding fragment thereof comprising the variable regions set forth in the heavy and light chain amino acid sequences set forth in Table 10 for any one of Ab54, Ab55, Ab56, Ab57, Ab58, Ab61 , Ab66, Ab67, Ab68, or Ab69.
  • the foregoing methods and compositions include an lgG1 anti-CD1 17 antibody or antigen-binding fragment thereof comprising the variable regions set forth in the heavy and light chain amino acid sequences set forth in Table 10 for any one of Ab54, Ab55, Ab56, Ab57, Ab58, Ab61 , Ab66, Ab67, Ab68, or Ab69.
  • Fig. 1 demonstrates the measurement of binding by Bio-Layer Interferometry (BLI) of the indicated purified IgG (sensor-associated) to purified human CD1 17 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM and 1 1 nM as a function of time.
  • BLI Bio-Layer Interferometry
  • Figs. 2A and 2B graphically depict the results of in vitro cell proliferation assays that show the dose-dependent effect of each indicated antibody on the Stem Cell Factor (SCF)-dependent proliferation of human CD34+ bone marrow cells.
  • SCF Stem Cell Factor
  • Fig. 3 demonstrates the measurement of binding by Bio-Layer Interferometry (BLI) of the indicated purified IgG (sensor-associated) to purified human CD1 17 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM and 1 1 nM as a function of time.
  • BLI Bio-Layer Interferometry
  • Figs. 4A and 4B graphically depict the results of in vitro cell proliferation assays that show the dose-dependent effect of each indicated antibody on the Stem Cell Factor (SCF)-dependent proliferation of human CD34+ bone marrow cells.
  • Total live cell counts (Fig. 4A) or viable CD34+ CD90+ cell counts as determined by flow cytometry (Fig. 4B) (y-axis) in the presence of the 3100 mAb, HC-55/LC-55 IgG (Ab55 IgG) or control as a function of 3100 mAb, HC-55/LC-55 IgG (Ab55 IgG) or control concentration (x-axis) are depicted.
  • Figs. 4A Total live cell counts
  • Fig. 4B y-axis in the presence of the 3100 mAb, HC-55/LC-55 IgG (Ab55 IgG) or control as a function of 3100 mAb, HC-55/LC-55 IgG (Ab55
  • FIG. 5A and 5B demonstrate the measurement of binding by Bio-Layer Interferometry (BLI) of the indicated purified IgG (sensor-associated) to purified human CD1 17 ectodomain (R&D Systems #332-SR) at concentrations of 33.3 nM and 1 1 nM as a function of time.
  • BLI Bio-Layer Interferometry
  • the results for Ab54, Ab55, Ab56, Ab57, Ab68, and Ab61 are shown in Fig. 5A.
  • the results for Ab66, Ab67, Ab68, and Ab69 are shown in Fig. 5B.
  • Figs. 6A and 6B graphically depicts the results of in vitro cell proliferation assays that show the effect of each indicated antibody on the Stem Cell Factor (SCF)-dependent proliferation of human CD34+ bone marrow cells.
  • SCF Stem Cell Factor
  • y-axis Total live cell counts as determined by flow cytometry (y-axis) in the presence of the indicated antibody or control (CK6) as a function of antibody concentration (x-axis) are depicted.
  • the results for Ab54, Ab55, Ab56, Ab57, Ab58, and Ab61 are shown in Fig. 6A.
  • the results for Ab66, Ab67, Ab68, and Ab69 are shown in Fig. 6B.
  • Fig. 7A and 7B graphically depicts the results of in vitro cell proliferation assays that show the effect of each indicated antibody on the Stem Cell Factor (SCF)-dependent proliferation of human CD34+ bone marrow cells.
  • SCF Stem Cell Factor
  • Viable CD34+ CD90+ cell counts as determined by flow cytometry (y-axis) in the presence of the indicated antibody or control (CK6) as a function of antibody concentration (x-axis) are depicted.
  • the results for Ab54, Ab55, Ab56, Ab57, Ab58, and Ab61 are shown in Fig. 7A.
  • the results for Ab66, Ab67, Ab68, and Ab69 are shown in Fig. 7B.
  • Fig. 8 graphically depicts the results of an in vitro cross-blocking assay in which the binding of CK6 or human SCF (y-axis) to Ab55 or Ab67 associated -CD1 17 was assessed as a function of time (x-axis).
  • Figs. 9A demonstrates the binding of human or murine SCF to human CD1 17 ectodomain (y-axis) as a function of time (x-axis) as determined by BLI. Subsequently, as shown in Fig. 9B, an in vitro cross-blocking assay demonstrated the binding of human or murine SCF (y-axis) to Ab67 associated-CD1 17 as a function of time (x-axis).
  • Figs. 10A and 10B graphically depict the results of an in vitro internalization assay.
  • the percent of surface CD1 17 was assessed on human bone marrow CD34+ cells following 24 hours of incubation with hlgG1 , an antagonist antibody, a neutral antibody, or SCF (Fig. 10A).
  • the corresponding percent of surface IgG was also assessed as a function of time (Fig. 10B).
  • Figs. 11 A-11 F depicts chromatograms demonstrating the elution profile of the indicated antibody after 7 days (CK6 control (Fig. 1 1 A), HC-55/LC-55 (Ab55; Fig. 1 1 B), or HC-67/LC-67
  • Fig. 12 graphically depicts electropherograms showing the charge heterogeneity profile of acidic variants present in the indicated antibody under the indicated incubation conditions (x-axis) as determined by capillary electrophoresis.
  • Figs. 13A and 13B depict multiple sequence alignments of the heavy chain variable regions (VH; Fig. 13A) and the light chain variable regions (LH; Fig. 13B) of antagonist antibodies Ab54, Ab55, Ab66, and Ab67. The CDRs of each variable region are indicated.
  • Figs. 14A and 14B depict multiple sequence alignments of the heavy chain variable regions (VH; Fig. 14A) and the light chain variable regions (LH; Fig. 14B) of neutral antibodies Ab58 and Ab61 .
  • the CDRs of each variable region are indicated.
  • Figs. 15A and 15B depict multiple sequence alignments of the heavy chain variable regions (VH; Fig. 15A) and the light chain variable regions (LH; Fig. 15B) of neutral antibodies Ab66, Ab67, Ab68, and Ab69. The CDRs of each variable region are indicated.
  • Described herein are isolated anti-CD1 17 human antibodies that bind to human CD1 17.
  • the antibodies provided herein have many characteristics making them advantageous for therapy, including methods of conditioning human patients for stem cell transplantation.
  • antibodies disclosed herein cross react with rhesus CD1 17 and are able to internalize.
  • the antibodies described herein include both antagonist antibodies and neutral antibodies.
  • Anti-CD1 17 antibodies Antibody 54 (Ab54), Antibody 55 (Ab55), Antibody 56 (Ab56), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61 ), Antibody 66 (Ab66), Antibody 67 (Ab67), Antibody 68 (Ab68), and Antibody 69 (Ab69) which are each human anti-CD1 17 antibodies that specifically bind to the ectodomain of human CD1 17.
  • the binding regions of Ab54, Ab55, Ab56, Ab57, Ab58, Ab61 , Ab66, Ab67, Ab68, and Ab69 are described below, including in Table 10.
  • the anti-CD1 17 antibodies herein can be used to in methods to treat a variety of disorders, such as diseases of a cell type in the hematopoietic lineage, cancers, autoimmune diseases, metabolic disorders, and stem cell disorders, among others.
  • the compositions and methods described herein may (i) directly deplete a population of cells that give rise to a pathology, such as a population of cancer cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T- cells), and/or (ii) deplete a population of endogenous hematopoietic stem cells so as to promote the engraftment of transplanted hematopoietic stem cells by providing a niche to which the transplanted cells may home.
  • cancer cells e.g., leukemia cells
  • autoimmune cells e.g., autoreactive T- cells
  • the foregoing activities can be achieved by administration of an antibody, or antigen-binding fragment thereof, capable of binding an antigen (CD1 17) expressed by an endogenous disease-causing cell or a hematopoietic stem cell.
  • this administration can cause a reduction in the quantity of the cells that give rise to the pathology of interest.
  • this administration can cause the selective depletion of a population of endogenous hematopoietic stem cells, thereby creating a vacancy in the hematopoietic tissue, such as the bone marrow, that can subsequently be filled by transplanted, exogenous hematopoietic stem cells.
  • This selective depletion is also referred to as conditioning.
  • the invention is based in part on the discovery that antibodies, and antigen-binding fragments thereof, capable of binding CD1 17 (such as GNNK+ D1 17) can be administered to a patient to effect both of the above activities or as a conditioning agent.
  • Antibodies, or antigen-binding fragments thereof, that bind CD1 17 can be administered to a patient suffering from a cancer, such as leukemia, or autoimmune disease to directly deplete a population of cancerous cells or autoimmune cells, and can also be administered to a patient in need of hematopoietic stem cell transplant therapy in order to promote the survival and engraftment potential of transplanted hematopoietic stem cells.
  • Engraftment of hematopoietic stem cell transplants due to the administration of anti-CD1 17 antibodies, or antigen-binding fragments thereof, can manifest in a variety of empirical measurements. For instance, engraftment of transplanted hematopoietic stem cells can be evaluated by assessing the quantity of competitive repopulating units (CRU) present within the bone marrow of a patient following administration of an antibody or antigen-binding fragment thereof capable of binding CD1 17 and subsequent administration of a hematopoietic stem cell transplant.
  • CRU competitive repopulating units
  • a reporter gene such as an enzyme that catalyzes a chemical reaction yielding a fluorescent, chromophoric, or luminescent product
  • hematopoietic stem cells have been transfected and subsequently monitoring the corresponding signal in a tissue into which the hematopoietic stem cells have homed, such as the bone marrow.
  • a tissue into which the hematopoietic stem cells have homed such as the bone marrow.
  • Engraftment can also be determined by measuring white blood cell counts in peripheral blood during a post-transplant period, and/or by measuring recovery of marrow cells by donor cells in a bone marrow aspirate sample.
  • the sections that follow provide a description of antibodies, and antigen-binding fragments thereof, that can be administered to a patient, such as a patient suffering from a cancer or autoimmune disease, or a patient in need of hematopoietic stem cell transplant therapy in order to promote engraftment of hematopoietic stem cell grafts, as well as methods of administering such therapeutics to a patient (e.g., prior to hematopoietic stem cell transplantation).
  • the term “about” refers to a value that is within 10% above or below the value being described.
  • the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad- specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab', F(ab') 2 , Fab, Fv, rlgG, and scFv fragments.
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab') 2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.
  • the antibodies of the present invention are generally isolated or recombinant.
  • isolated when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. For instance, an isolated antibody that specifically binds to CD1 17 is substantially free of antibodies that specifically bind antigens other than CD1 17.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragments can be, for example, a Fab, F(ab') 2 , scFv, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term
  • antigen-binding fragment of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H 1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including V H and V L domains; (vi) a dAb fragment that consists of a V H domain (see, e.g., Ward et al., Nature 341 :544-546, 1989); (vii) a dAb which consists of a V H or a V L domain; (viii) an isolated complementarity determining region (CDR); and (ix)
  • single chain Fv single chain Fv
  • scFv single chain Fv
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.
  • anti-CD1 17 antibody or “an antibody that binds to CD1 17” refers to an antibody that is capable of binding CD1 17 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD1 17.
  • the amino acid sequences of the two main isoforms of human CD1 17 are provided in SEQ ID NO: 145 (isoform 1 ) and SEQ ID NO: 146 (isoform 2).
  • bispecific antibody refers to, for example, a monoclonal, often a human or humanized antibody that is capable of binding at least two different antigens.
  • hematopoietic stem cell surface antigen or another cell surface protein such as a receptor or receptor subunit involved in a signal transduction pathway that potentiates cell growth, among others.
  • CDR complementarity determining region
  • FRs framework regions
  • the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions.
  • variable domains of native heavy and light chains each contain four framework regions that primarily adopt a ⁇ -sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the ⁇ - sheet structure.
  • the CDRs in each chain are held together in close proximity by the framework regions in the order FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD., 1987).
  • numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated (although any antibody numbering scheme, including, but not limited to IMGT and Chothia, can be utilized).
  • condition refers to processes by which a patient is prepared for receipt of a transplant containing hematopoietic stem cells. Such procedures promote the engraftment of a hematopoietic stem cell transplant (for instance, as inferred from a sustained increase in the quantity of viable hematopoietic stem cells within a blood sample isolated from a patient following a conditioning procedure and subsequent hematopoietic stem cell transplantation.
  • a patient may be
  • hematopoietic stem cells such as CD1 17 (e.g., GNNK+ CD1 17).
  • Administration of an antibody, or antigen-binding fragment thereof, capable of binding one or more of the foregoing antigens to a patient in need of hematopoietic stem cell transplant therapy can promote the engraftment of a hematopoietic stem cell graft, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating a vacancy filled by an exogenous hematopoietic stem cell transplant.
  • CRU competitive repopulating unit
  • the term "donor” refers to a human or animal from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient.
  • the one or more cells may be, for example, a population of hematopoietic stem cells.
  • the term "diabody” refers to a bivalent antibody containing two polypeptide chains, in which each polypeptide chain includes V H and V L domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of V H and V L domains on the same peptide chain. This configuration forces each domain to pair with a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of V H and V L domains on the same peptide chain. This configuration forces each domain to pair with a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of V H and V L domains on the same peptide chain. This configuration forces each domain to pair with a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of V H and V L domains on the same peptid
  • the term "triabody” refers to trivalent antibodies containing three peptide chains, each of which contains one V H domain and one V L domain joined by a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain.
  • a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain.
  • peptides configured in this way typically trimerize so as to position the V H and V L domains of neighboring peptide chains spatially proximal to one another (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).
  • a “dual variable domain immunoglobulin” refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent (see, for example, Gu et al., Meth. Enzymol.,
  • the term "endogenous” describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell,
  • a hematopoietic stem cell or a cell of hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell,
  • the term "engraftment potential” is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate a tissue, whether such cells are naturally circulating or are provided by transplantation.
  • the term encompasses all events surrounding or leading up to engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest.
  • the engraftment efficiency or rate of engraftment can be evaluated or quantified using any clinically acceptable parameter as known to those of skill in the art and can include, for example, assessment of competitive repopulating units (CRU); incorporation or expression of a marker in tissue(s) into which stem cells have homed, colonized, or become engrafted; or by evaluation of the progress of a subject through disease progression, survival of hematopoietic stem and progenitor cells, or survival of a recipient.
  • Engraftment can also be determined by measuring white blood cell counts in peripheral blood during a post-transplant period. Engraftment can also be assessed by measuring recovery of marrow cells by donor cells in a bone marrow aspirate sample.
  • exogenous describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell,
  • a hematopoietic stem cell or a cell of hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell,
  • Exogenous substances include those that are provided from an external source to an organism or to cultured matter extracted therefrom.
  • frame region includes amino acid residues that are adjacent to the CDRs of an antibody or antigen-binding fragment thereof.
  • FW region residues may be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.
  • Fc refers to the portion of an IgG antibody that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
  • the Fc region comprises the C-terminal half of two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and binding sites for complement and Fc receptors, including the FcRn receptor.
  • An Fc region contains the second constant domain CH2 (e.g., residues at EU positions 231 -340 of lgG1 ) and the third constant domain CH3 (e.g., residues at EU positions 341 -447 of human lgG1 ).
  • the Fc region includes the "lower hinge region” (e.g., residues at EU positions 233- 239 of lgG1 ).
  • Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of positions in Fc domains, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and thus slight differences between the sequences presented in the instant application and sequences known in the art can exist.
  • a "wild type IgG Fc domain” or "WT IgG Fc domain” refers to any naturally occurring IgG Fc region (i.e., any allele).
  • sequences of the heavy chains of human lgG1 , lgG2, lgG3 and lgG4 can be found in a number of sequence databases, for example, at the Uniprot database (www.uniprot.org) under accession numbers P01857
  • modified Fc region or “variant Fc region” as used herein refers to an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc region.
  • an intact antibody comprises two heavy chains each comprising a variable region, a constant region and an Fc region, and two light chains each comprising a variable region and a constant region. More specifically, an intact IgG comprises two light chains each comprising a light chain variable region (VL) and a light chain constant region (CL), and comprises two heavy chains each comprising a heavy chain variable region (VH) and three heavy chain constant regions (CH1 , CH2, and CH3). CH2 and CH3 represent the Fc region of the heavy chain.
  • frame region includes amino acid residues that are adjacent to the CDRs of an antibody or antigen-binding fragment thereof.
  • FW region residues may be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.
  • HSCs hematopoietic stem cells
  • granulocytes e.g., promyelocytes, neutrophils, eosinophils, basophils
  • erythrocytes e.g., reticulocytes, erythrocytes
  • thrombocytes e.g., megakaryoblasts, platelet producing megakaryocytes, platelets
  • monocytes e.g., monocytes, macrophages
  • dendritic cells e.g., NK cells, B-cells and T-cells.
  • Such cells may include CD34 + cells.
  • CD34 + cells are immature cells that express the CD34 cell surface marker.
  • CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above, whereas in mice, HSCs are CD34-.
  • HSCs also refer to long term repopulating HSCs (LT-HSC) and short term repopulating HSCs (ST- HSC).
  • LT-HSCs and ST-HSCs are differentiated, based on functional potential and on cell surface marker expression.
  • human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD1 1 B, CD19, CD20, CD56, CD235A).
  • bone marrow LT-HSCs are CD34-, SCA-1 +, C- kit+, CD135-, Slamfl/CD150+, CD48-, and lin- (negative for mature lineage markers including Ter1 19, CD1 1 b, Gr1 , CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are CD34+, SCA-1 +, C- kit+, CD135-, Slamfl/CD150+, and lin- (negative for mature lineage markers including Ter1 19, CD1 1 b, Gr1 , CD3, CD4, CD8, B220, IL7ra).
  • ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions.
  • LT-HSC have greater self- renewal potential (i.e., they survive throughout adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time, and do not possess serial transplantation potential). Any of these HSCs can be used in the methods described herein.
  • ST-HSCs are particularly useful because they are highly proliferative and thus, can more quickly give rise to differentiated progeny.
  • hematopoietic stem cell functional potential refers to the functional properties of hematopoietic stem cells which include 1 ) multi-potency (which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing
  • multi-potency which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing
  • monocytes e.g., monocytes, macrophages
  • dendritic cells e.g., microglia, osteoclasts
  • lymphocytes e.g., NK cells, B-cells and T-cells
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • a human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • a human antibody can be produced in a human cell (for example, by recombinant expression) or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (such as heavy chain and/or light chain) genes.
  • a human antibody when a human antibody is a single chain antibody, it can include a linker peptide that is not found in native human antibodies.
  • an Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, for example, PCT Publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Patent Nos. 5,413,923; 5,625,126;
  • a “humanized” antibody refers to an antibody that contains minimal sequences derived from non-human immunoglobulin.
  • "humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. All or substantially all of the FW regions may also be those of a human immunoglobulin sequence.
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • patients that are "in need of" a hematopoietic stem cell transplant include patients that exhibit a defect or deficiency in one or more blood cell types, as well as patients having a stem cell disorder, autoimmune disease, cancer, or other pathology described herein.
  • Hematopoietic stem cells generally exhibit 1 ) multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes,
  • neutrophils neutrophils, eosinophils, basophils
  • erythrocytes e.g., reticulocytes, erythrocytes
  • thrombocytes e.g., megakaryoblasts, platelet producing megakaryocytes, platelets
  • monocytes e.g., monocytes, macrophages
  • dendritic cells microglia, osteoclasts, and lymphocytes
  • lymphocytes e.g., NK cells, B-cells and T-cells
  • 2) self-renewal and can thus give rise to daughter cells that have equivalent potential as the mother cell, and 3) the ability to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.
  • Hematopoietic stem cells can thus be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re- constitute the defective or deficient population of cells in vivo.
  • the patient may be suffering from cancer, and the deficiency may be caused by administration of a chemotherapeutic agent or other medicament that depletes, either selectively or non-specifically, the cancerous cell population.
  • the patient may be suffering from a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • a hemoglobinopathy e.g., a non-malignant hemoglobinopathy
  • the subject may be one that is suffering from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
  • ADA SCID adenosine deaminase severe combined immunodeficiency
  • the subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder.
  • the subject may have or be affected by a malignancy, such as neuroblastoma or a hematologic cancer.
  • the subject may have a leukemia, lymphoma, or myeloma.
  • the subject has acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the subject has myelodysplastic syndrome.
  • the subject has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1 diabetes, or another autoimmune pathology described herein.
  • the subject is in need of chimeric antigen receptor T-cell (CART) therapy.
  • the subject has or is otherwise affected by a metabolic storage disorder.
  • the subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, metachromatic leukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as
  • a patient "in need of" a hematopoietic stem cell transplant may one that is or is not suffering from one of the foregoing pathologies, but nonetheless exhibits a reduced level (e.g., as compared to that of an otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T- lymphocytes, and B- lymphocytes.
  • endogenous cell types within the hematopoietic lineage such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophil
  • FACS fluorescence activated cell sorting
  • the term "monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a “neutral antibody” refers to an antibody, or an antigen binding fragment thereof, that is not capable of significantly neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a particular or specified target (e.g., CD1 17), including the binding of receptors to ligands or the interactions of enzymes with substrates.
  • a neutral anti-CD1 17 antibody is an anti-CD1 17 antibody that does not substantially inhibit SCF-dependent cell proliferation and does not cross block SCF binding to CD1 17.
  • An example of a neutral antibody is Ab67 (or an antibody having the binding regions of Ab67).
  • an "antagonist" anti-CD1 17 antibody inhibits SCF-dependent proliferation and is able to cross block SCF binding to CD1 17.
  • An example of an antagonist antibody is Ab55 (or an antibody having the binding regions of Ab55).
  • the term "recipient” refers to a patient that receives a transplant, such as a transplant containing a population of hematopoietic stem cells.
  • a transplant such as a transplant containing a population of hematopoietic stem cells.
  • administered to a recipient may be, e.g., autologous, syngeneic, or allogeneic cells.
  • sample refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells) taken from a subject.
  • a specimen e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells
  • scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
  • scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (V L ) (e.g., CDR-L1 , CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (V H ) (e.g., CDR-H1 , CDR-H2, and/or CDR-H3) separated by a linker.
  • V L variable region of an antibody light chain
  • V H variable region of an antibody heavy chain
  • the linker that joins the V L and V H regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
  • linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (for example, linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (for example, linkers containing glycosylation sites).
  • linkers containing D-amino acids for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues) so as to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.
  • telomere binding refers to the ability of an antibody to recognize and bind to a specific protein structure (epitope) rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody. By way of example, an antibody “binds specifically" to a target if the antibody, when labeled, can be competed away from its target by the corresponding non-labeled antibody.
  • an antibody specifically binds to a target, e.g., CD1 17, if the antibody has a K D for the target of at least about 10 "4 M, 10 "5 M, 10 “6 M, 10 “7 M, 10 “8 M, 10 “9 M, 10 "10 M, 10 "11 M, 10 "12 M, or less (less meaning a number that is less than 10 "12 , e.g. 10 "13 ).
  • K D (M) is determined according to standard bio-layer interferometery (BLI).
  • K off (1 /s) is determined according to standard bio-layer interferometery (BLI).
  • the antibody may be capable of specifically binding to two or more antigens which are related in sequence.
  • an antibody can specifically bind to both human and a non-human (e.g., mouse or non-human primate) orthologs of CD1 17.
  • the terms “subject” and “patient” refer to an organism, such as a human, that receives treatment for a particular disease or condition as described herein.
  • a patient such as a human patient, may receive treatment prior to hematopoietic stem cell transplant therapy in order to promote the engraftment of exogenous hematopoietic stem cells.
  • stem cell disorder broadly refers to any disease, disorder, or condition that may be treated or cured by conditioning a subject's target tissues, and/or by ablating an endogenous stem cell population in a target tissue (e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue) and/or by engrafting or transplanting stem cells in a subject's target tissues.
  • a target tissue e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue
  • stem cells in a subject's target tissues e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue
  • Type I diabetes has been shown to be cured by hematopoietic stem cell transplant and may benefit from conditioning in accordance with the compositions and methods described herein.
  • Additional disorders that can be treated using the compositions and methods described herein include, without limitation, sickle cell anemia, thalassemias, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
  • Additional diseases that may be treated using the patient conditioning and/or hematopoietic stem cell transplant methods described herein include inherited blood disorders (e.g., sickle cell anemia) and autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease.
  • Additional diseases that may be treated using the conditioning and/or transplantation methods described herein include a malignancy, such as a neuroblastoma or a hematologic cancer, such as leukemia, lymphoma, and myeloma.
  • the cancer may be acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non- Hodgkin's lymphoma.
  • Additional diseases treatable using the conditioning and/or transplantation methods described herein include myelodysplastic syndrome.
  • the subject has or is otherwise affected by a metabolic storage disorder.
  • the subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,
  • sphingolipidoses metachromatic leukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains to pathologies that may be treated by administration of hematopoietic stem cell transplant therapy.
  • IgM hyper immunoglobulin M
  • transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like.
  • treat refers to reducing the severity and/or frequency of disease symptoms, eliminating disease symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by disease.
  • beneficial or desired clinical results include, but are not limited to, promoting the engraftment of exogenous hematopoietic cells in a patient following antibody conditioning therapy as described herein and subsequent hematopoietic stem cell transplant therapy.
  • Additional beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem cell transplant following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to the patient.
  • Beneficial results of therapy described herein may also include an increase in the cell count or relative concentration of one or more cells of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T- lymphocyte, or B-lymphocyte, following conditioning therapy and subsequent hematopoietic stem cell transplant therapy.
  • hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T- lymphocyte, or B-lymphocyte, following conditioning
  • Additional beneficial results may include the reduction in quantity of a disease-causing cell population, such as a population of cancer cells (e.g., CD1 17+ leukemic cells) or autoimmune cells (e.g., CD1 17+ autoimmune lymphocytes, such as a CD1 17+ T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen).
  • a disease-causing cell population such as a population of cancer cells (e.g., CD1 17+ leukemic cells) or autoimmune cells (e.g., CD1 17+ autoimmune lymphocytes, such as a CD1 17+ T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen).
  • cancer cells e.g., CD1 17+ leukemic cells
  • autoimmune cells e.g., CD1 17+ autoimmune lymphocytes, such as a CD1 17+ T-cell that expresses a T-cell receptor that cross-reacts with
  • the term preventing refers to the ability of the skilled artisan to identify a population that is susceptible to disorders, such that administration of the compounds of the present invention may occur prior to onset of a disease. The term does not imply that the disease state is completely avoided.
  • variants and “derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein.
  • a variant or derivative of a compound, peptide, protein, or other substance described herein may retain or improve upon the biological activity of the original material.
  • vector includes a nucleic acid vector, such as a plasmid, a DNA vector, a plasmid, a RNA vector, virus, or other suitable replicon.
  • Expression vectors described herein may contain a polynucleotide sequence as well as, for example, additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • kits for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.
  • Anti-CD117 Antibodies include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.
  • the present invention is based in part on the discovery that antibodies, or antigen-binding fragments thereof, capable of binding CD1 17, such as GNNK+ CD1 17, can be used as therapeutic agents to (i) treat cancers and autoimmune diseases characterized by CD1 17+ cells and (ii) promote the engraftment of transplanted hematopoietic stem cells in a patient in need of transplant therapy.
  • These therapeutic activities can be caused, for instance, by the binding of isolated anti- CD1 17 antibodies, antigen-binding fragments thereof, that bind to CD1 17 (e.g., GNNK+ CD1 17) expressed on the surface of a cell, such as a cancer cell, autoimmune cell, or hematopoietic stem cell and subsequently inducing cell death.
  • transplanted hematopoietic stem cells may successfully engraft in a patient, such as human patient suffering from a stem cell disorder described herein.
  • Antibodies and antigen-binding fragments capable of binding human CD1 17 can be used in conjunction with the compositions and methods described herein in order to condition a patient for
  • CD1 17 Polymorphisms affecting the coding region or extracellular domain of CD1 17 in a significant percentage of the population are not currently well- known in non-oncology indications.
  • Two of the CD1 17 isoforms are located on the intracellular domain of the protein, and two are present in the external juxtamembrane region.
  • the two extracellular isoforms, GNNK+ and GNNK- differ in the presence (GNNK+) or absence (GNNK-) of a 4 amino acid sequence.
  • the GNNK+ isoform can be used as an immunogen in order to generate antibodies capable of binding CD1 17, as antibodies generated against this isoform will be inclusive of the GNNK+ and GNNK- proteins.
  • the amino acid sequences of human CD1 17 isoforms 1 and 2 are described in SEQ ID Nos: 145 and 146, respectively.
  • anti-human CD1 17 (hCD1 17) antibodies disclosed herein are able to bind to both isoform 1 and isoform 2 of human CD1 17.
  • yeast library screen of human antibodies was performed to identify novel anti-CD1 17 antibodies, and fragments thereof, having diagnostic and therapeutic use.
  • Antibody 54 (Ab54), Antibody 55 (Ab55), Antibody 56 (Ab56), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61 ), Antibody 66 (Ab66), Antibody 67 (Ab67), Antibody 68 (Ab68), and Antibody 69 (Ab69) were human antibodies that were identified in this screen. These antibodies cross react with human CD1 17 and rhesus CD1 17. Further, these antibodies disclosed herein are able to bind to both isoforms of human CD1 17, i.e., isoform 1 (SEQ ID NO: 145) and isoform 2 (SEQ ID NO: 146).
  • anti-CD1 17 antibodies Ab54, Ab55, Ab56, Ab57, Ab58, Ab61 , Ab66, Ab67, Ab68, and Ab69 are described in Table 10. Included in the invention are human anti-CD1 17 antibodies comprising the CDRs as set forth in Table 10, as well as human anti-CD1 17 antibodies comprising the variable regions set forth in Table 10.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 55.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 55 i.e., Ab55
  • the VH CDR domain amino acid sequences of Antibody 55 are set forth in SEQ ID NO: 21 (VH CDR1 ); SEQ ID NO: 22 (VH CDR2), and SEQ ID NO: 23 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 55 is described in SEQ ID NO: 20 (see Table 10).
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 21 , 22, and 23, and a light chain variable region CDR set as set forth in SEQ ID Nos: 24, 25, and 26.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 20, and a heavy chain variable region as set forth in SEQ ID NO: 19.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 54.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 54 i.e., Ab54
  • the VH CDR domain amino acid sequences of Antibody 54 are set forth in SEQ ID NO: 31 (VH CDR1 ); SEQ ID NO: 32 (VH CDR2), and SEQ ID NO: 33 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 54 is described in SEQ ID NO: 30 (see Table 10).
  • VL CDR domain amino acid sequences of Antibody 54 are set forth in SEQ ID NO: 34 (VL CDR1 ); SEQ ID NO: 35 (VL CDR2), and SEQ ID NO: 36 (VL CDR3).
  • the heavy chain constant region of Antibody 54 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 54 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 31 , 32, and 33, and a light chain variable region CDR set as set forth in SEQ ID Nos: 34, 35, and 36.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 30, and a heavy chain variable region as set forth in SEQ ID NO: 29.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 56.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 56 i.e., Ab56
  • the VH CDR domain amino acid sequences of Antibody 56 are set forth in SEQ ID NO: 41 (VH CDR1 ); SEQ ID NO: 42 (VH CDR2), and SEQ ID NO: 43 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 56 is described in SEQ ID NO: 40 (see Table 10).
  • VL CDR domain amino acid sequences of Antibody 56 are set forth in SEQ ID NO: 44 (VL CDR1 ); SEQ ID NO: 45 (VL CDR2), and SEQ ID NO: 46 (VL CDR3).
  • the heavy chain constant region of Antibody 56 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 56 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 41 , 42, and 43, and a light chain variable region CDR set as set forth in SEQ ID Nos: 44, 45, and 46.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 40, and a heavy chain variable region as set forth in SEQ ID NO: 39.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 57.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 57 i.e., Ab57
  • the VH CDR domain amino acid sequences of Antibody 57 are set forth in SEQ ID NO: 51 (VH CDR1 ); SEQ ID NO: 52 (VH CDR2), and SEQ ID NO: 53 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 57 is described in SEQ ID NO: 50 (see Table 10).
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 51 , 52, and 53, and a light chain variable region CDR set as set forth in SEQ ID Nos: 54, 55, and 56.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 50, and a heavy chain variable region as set forth in SEQ ID NO: 49.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 58.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 58 i.e., Ab58
  • SEQ ID NO: 59 see Table 10
  • the VH CDR domain amino acid sequences of Antibody 58 are set forth in SEQ ID NO: 61 (VH CDR1 ); SEQ ID NO: 62 (VH CDR2), and SEQ ID NO: 63 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 58 is described in SEQ ID NO: 60 (see Table 10).
  • VL CDR domain amino acid sequences of Antibody 58 are set forth in SEQ ID NO: 64 (VL CDR1 ); SEQ ID NO: 65 (VL CDR2), and SEQ ID NO: 66 (VL CDR3).
  • the heavy chain constant region of Antibody 58 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 58 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 61 , 62, and 63, and a light chain variable region CDR set as set forth in SEQ ID Nos: 64, 65, and 66.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 60, and a heavy chain variable region as set forth in SEQ ID NO: 59.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 61 .
  • the heavy chain variable region (VH) amino acid sequence of Antibody 61 i.e., Ab61
  • the VH CDR domain amino acid sequences of Antibody 61 are set forth in SEQ ID NO: 71 (VH CDR1 ); SEQ ID NO: 72 (VH CDR2), and SEQ ID NO: 73 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 61 is described in SEQ ID NO: 70 (see Table 10).
  • Antibody 61 are set forth in SEQ ID NO: 74 (VL CDR1 ); SEQ ID NO: 75 (VL CDR2), and SEQ ID NO: 76 (VL CDR3).
  • the heavy chain constant region of Antibody 61 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 61 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 71 , 72, and 73, and a light chain variable region CDR set as set forth in SEQ ID Nos: 74, 75, and 76.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 70, and a heavy chain variable region as set forth in SEQ ID NO: 69.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 66.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 66 i.e., Ab66
  • the VH CDR domain amino acid sequences of Antibody 66 are set forth in SEQ ID NO: 81 (VH CDR1 ); SEQ ID NO: 82 (VH CDR2), and SEQ ID NO: 83 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 66 is described in SEQ ID NO: 80 (see Table 10).
  • VL CDR domain amino acid sequences of Antibody 66 are set forth in SEQ ID NO: 84 (VL CDR1 ); SEQ ID NO: 85 (VL CDR2), and SEQ ID NO: 86 (VL CDR3).
  • the heavy chain constant region of Antibody 66 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 66 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 81 , 82, and 83, and a light chain variable region CDR set as set forth in SEQ ID Nos: 84, 85, and 86.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 80, and a heavy chain variable region as set forth in SEQ ID NO: 79.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 67.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 67 is set forth in SEQ ID NO: 9 (see Table 2).
  • the VH CDR domain amino acid sequences of Antibody 67 are set forth in SEQ ID NO 1 1 (VH CDR1 ); SEQ ID NO: 12 (VH CDR2), and SEQ ID NO: 13 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 67 is described in SEQ ID NO: 10 (see Table 2).
  • the VL CDR domain amino acid sequences of Antibody 67 are set forth in SEQ ID NO 14 (VL CDR1 ); SEQ ID NO: 15 (VL CDR2), and SEQ ID NO: 16 (VL CDR3).
  • the full length heavy chain (HC) of Antibody 67 is set forth in SEQ ID NO: 1 10
  • the full length heavy chain constant region of Antibody 67 is set forth in SEQ ID NO: 122.
  • the light chain (LC) of Antibody 67 is set forth in SEQ ID NO: 109.
  • the light chain constant region of Antibody 67 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen- binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 1 1 , 12, and 13, and a light chain variable region CDR set as set forth in SEQ ID Nos: 14, 15, and 16.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 9, and a heavy chain variable region as set forth in SEQ ID NO: 10.
  • an anti-CD1 17 antibody comprises a heavy chain comprising SEQ ID NO: 1 10 and a light chain comprising SEQ ID NO: 109.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 68.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 68 i.e., Ab68
  • SEQ ID NO: 89 see Table 10
  • the VH CDR domain amino acid sequences of Antibody 68 are set forth in SEQ ID NO: 91 (VH CDR1 ); SEQ ID NO: 92 (VH CDR2), and SEQ ID NO: 93 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 68 is described in SEQ ID NO: 90 (see Table 10).
  • VL CDR domain amino acid sequences of Antibody 68 are set forth in SEQ ID NO: 94 (VL CDR1 ); SEQ ID NO: 95 (VL CDR2), and SEQ ID NO: 96 (VL CDR3).
  • the heavy chain constant region of Antibody 68 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 68 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 91 , 92, and 93, and a light chain variable region CDR set as set forth in SEQ ID Nos: 94, 95, and 96.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 90, and a heavy chain variable region as set forth in SEQ ID NO: 89.
  • the invention provides an anti-CD1 17 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 69.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 69 i.e., Ab69
  • SEQ ID NO: 99 see Table 10
  • the VH CDR domain amino acid sequences of Antibody 69 are set forth in SEQ ID NO: 101 (VH CDR1 ); SEQ ID NO: 102 (VH CDR2), and SEQ ID NO: 103 (VH CDR3).
  • Antibody 69 is described in SEQ ID NO: 100 (see Table 10).
  • the VL CDR domain amino acid sequences of Antibody 69 are set forth in SEQ ID NO: 104 (VL CDR1 ); SEQ ID NO: 105 (VL CDR2), and SEQ ID NO: 106 (VL CDR3).
  • the heavy chain constant region of Antibody 69 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 69 is set forth in SEQ ID NO: 121 .
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 101 , 102, and 103, and a light chain variable region CDR set as set forth in SEQ ID Nos: 104, 105, and 106.
  • an anti-CD1 17 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 100, and a heavy chain variable region as set forth in SEQ ID NO: 99.
  • anti-CD1 17 antibodies described herein are neutral antibodies, in that the antibodies do not substantially inhibit CD1 17 activity on a CD1 17 expressing cell.
  • Neutral antibodies can be identified using, for example, an in in vitro stem cell factor (SCF)-dependent cell proliferation assay (see, e.g., Example 1 1 described herein).
  • SCF stem cell factor
  • a neutral CD1 17 antibody will not kill CD34+ cells that are dependent on SCF to divide, as a neutral antibody will not block SCF from binding to CD1 17 such as to inhibit CD1 17 activity.
  • Neutral antibodies can also be used for diagnostic purposes, given their ability to specifically bind to human CD1 17,.
  • Examples of neutral anti-CD1 17 antibodies include Ab58, Ab61 , Ab66, Ab67, Ab68, and Ab69.
  • a comparison of the amino acid sequences of the CDRs of neutral, anti-CD1 17 antibody CDRs reveals consensus sequences among two groups of neutral antibodies identified.
  • a comparison of the heavy and light chain variable regions of Ab58 and Ab61 is described in Figs. 14A and 14B, respectively.
  • Ab58 and Ab61 share the same light chain CDRs and the same HC CDR 3, with slight variations in the HC CDR1 and HC CDR2.
  • Consensus sequences for the HC CDR1 and HC CDR2 are described in SEQ ID Nos: 133 and 134.
  • Ab66, Ab67, Ab68, and Ab69 are also neutral antibodies.
  • Fig. 15A and Fig. 15B The heavy and light chain variable regions of these antibodies are described in Fig. 15A and Fig. 15B, respectively. While Ab66, Ab67, Ab68, and Ab69 share the same light chain CDRs and the same HC CDR3, these antibodies have variability within their HC CDR1 and HC CDR2 regions. Consensus sequences for these antibodies in the HC CDR1 and HC CDR2 regions are provided in SEQ ID Nos: 139 and 140, respectively.
  • Antagonist antibodies are also provided herein, including Ab54, Ab55, Ab56, and Ab57.
  • a comparison of the variable heavy and light chain amino acid sequences for these antibodies is provided in Figs. 13A and 13, respectively. While Ab54, Ab55, Ab56, and Ab57 share the same light chain CDRs and the same HC CDR3, these antibodies have variability within their HC CDR1 and HC CDR2 regions. Consensus sequences for these antibodies in the HC CDR1 and CDR2 regions are provided in SEQ ID Nos: 127 and 128, respectively.
  • the anti-CD1 17 antibodies described herein can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multiple chain or single chain antibodies, and/or binding fragments that specifically bind human CD1 17, including but not limited to Fab, Fab', (Fab')2, Fv), scFv (single chain Fv), surrobodies (including surrogate light chain construct), single domain antibodies, camelized antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g., lgA1 or lgA2), IgD, IgE, IgG (e.g.
  • the anti-CD1 17 antibody is an IgG (e.g. lgG1 , lgG2, lgG3 or lgG4).
  • Antibodies for use in conjunction with the methods described herein include variants of those antibodies described above, such as antibody fragments that contain or lack an Fc domain, as well as humanized variants of non-human antibodies described herein and antibody-like protein scaffolds (e.g., 10 Fn3 domains) containing one or more, or all, of the CDRs or equivalent regions thereof of an antibody, or antibody fragment, described herein.
  • antibody-like protein scaffolds e.g. 10 Fn3 domains
  • Exemplary antigen-binding fragments of the foregoing antibodies include a dual-variable immunoglobulin domain, a single- chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab') 2 molecule, and a tandem di-scFv, among others.
  • scFv single-chain Fv molecule
  • anti-CD1 17 antibodies comprising one or more radiolabeled amino acids are provided.
  • a radiolabeled anti-CD1 17 antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature).
  • Nonlimiting examples of labels for polypeptides include, but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251, 131 1, and 186Re.
  • Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681 ,581 , U.S. Pat. No. 4,735,210, U.S. Pat. No. 5,101 ,827, U.S. Pat. No. 5,102,990 (U.S. RE35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902.
  • a radioisotope may be conjugated by a chloramine T method.
  • anti-CD1 17 antibodies or binding fragments described herein may also include modifications and/or mutations that alter the properties of the antibodies and/or fragments, such as those that increase half-life, increase or decrease ADCC, etc., as is known in the art.
  • the anti-CD1 17 antibody, or binding fragment thereof comprises a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule has an altered affinity for an FcgammaR.
  • Certain amino acid positions within the Fc region are known through crystallography studies to make a direct contact with FcvR. Specifically amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino acids 327-332 (F/G) loop, (see Sondermann et al., 2000 Nature, 406: 267-273).
  • the anti-CD1 17 antibodies described herein may comprise variant Fc regions comprising modification of at least one residue that makes a direct contact with an Fey R based on structural and crystallographic analysis.
  • the Fc region of the anti-CD1 17 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1 , MD (1991 ), expressly incorporated herein by references.
  • the "EU index as in Kabat” refers to the numbering of the human lgG1 EU antibody.
  • the EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.)
  • the Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. In some
  • the Fc region of the anti-CD1 17 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a L234A mutation. In some embodiments, the Fc region of the anti- CD1 17 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises a L234A and L235A mutation . In a further embodiment, the Fc region comprises a D265C, L234A, and L235A mutation.
  • a variant IgG Fc domain comprises one or more amino acid substitutions resulting in decreased or ablated binding affinity for an Fc.gamma.R and/or C1 q as compared to the wild type Fc domain not comprising the one or more amino acid substitutions.
  • Fc binding interactions are essential for a variety of effector functions and downstream signaling events including, but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and
  • an antibody comprising a modified Fc region (e.g., comprising a L234A, L235A, and a D265C mutation) has substantially reduced or abolished effector functions.
  • Affinity to an Fc region can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol.
  • radioimmunoassay RIA
  • a surface plasmon resonance assay or other mechanism of kinetics-based assay e.g., BIACORETM analysis or OctetTM analysis (forteBIO)
  • FRET fluorescence resonance energy transfer
  • gel electrophoresis e.g., gel filtration
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis.
  • Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.
  • the antibodies of the invention may be further engineered to further modulate antibody half-life by introducing additional Fc mutations, such as those described for example in (Dall'Acqua et al. (2006) J Biol Chem 281 : 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol 176: 346-56), (Shields et al. (2001 ) J Biol Chem 276: 6591 -604), (Petkova et al. (2006) Int Immunol 18: 1759- 69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94), (Vaccaro et al. (2005) Nat
  • the Fc region comprises a mutation resulting in a decrease in half life.
  • An antibody having a short half life may be advantageous in certain instances where the antibody is expected to function as a short-lived therapeutic, e.g., the conditioning step described herein where the antibody is administered followed by HSCs. Ideally, the antibody would be substantially cleared prior to delivery of the HSCs, which also generally express CD1 17 but are not the target of the anti-CD1 17 antibody, unlike the endogenous stem cells.
  • the Fc regions comprise a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is an H435A mutation.
  • the anti-CD1 17 antibody described herein has a half life of equal to or less than 24 hours, a half life of equal to or less than 22 hours, a half life of equal to or less than 20 hours, a half life of equal to or less than 18 hours, a half life of equal to or less than 16 hours, a half life of equal to or less than 14 hours, equal to or less than 13 hours, equal to or less than 12 hours, or equal to or less than 1 1 hours.
  • the Fc region comprises two or more mutations that confer reduced half- life and greatly diminish or completely abolish an effector function of the antibody.
  • the Fc region comprises a mutation resulting in a decrease in half-life and a mutation of at least one residue that can make direct contact with an FcvR (e.g., as based on structural and crystallographic analysis).
  • the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation.
  • the Fc region comprises a H435A mutation and a D265C mutation.
  • the Fc region comprises a H435A mutation ,a L234A mutation ,a L235A mutation, and a D265C mutation.
  • the Fc region of Antibody 67 is modified to comprise a D265C mutation (e.g., SEQ ID NO: 1 1 1 ).
  • the Fc region of Antibody 67 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 1 12).
  • the Fc region of Antibody 67 is modified to comprise a D265C and H435A mutation (e.g., SEQ ID NO: 1 13).
  • the Fc region of Antibody 67 is modified to comprise a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO: 1 14).
  • the Fc region of Antibody 55 is modified to comprise a D265C mutation (e.g., SEQ ID NO: 1 17). In another embodiment, the Fc region of Antibody 55 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 1 18). In yet another embodiment, the Fc region of Antibody 55 is modified to comprise a D265C and H435A mutation (e.g., SEQ ID NO: 1 19). In a further embodiment, the Fc region of Antibody 55 is modified to comprise a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO: 120).
  • a D265C mutation e.g., SEQ ID NO: 1 17
  • the Fc region of Antibody 55 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 1 18).
  • the Fc region of Antibody 55 is
  • the Fc regions of any one of Antibody 54, Antibody 55, Antibody 56, Antibody 57, Antibody 58, Antibody 61 , Antibody 66, Antibody 67, Antibody 68, or Antibody 69 can be modified to comprise a D265C mutation (e.g., as in SEQ ID NO: 123); a D265C, L234A, and L235A mutation (e.g., as in SEQ ID NO: 124); a D265C and H435A mutation (e.g., as in SEQ ID NO: 125); or a D265C, L234A, L235A, and H435A mutation (e.g., as in SEQ ID NO: 126).
  • a D265C mutation e.g., as in SEQ ID NO: 123
  • a D265C, L234A, and L235A mutation e.g., as in SEQ ID NO: 124
  • D265C is an Fc variant with the aspartic acid (D) at EU position 265 substituted with cysteine (C) relative to the parent Fc domain.
  • D265C/L234A/L235A defines a variant Fc variant with substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parent Fc domain.
  • a variant can also be designated according to its final amino acid composition in the mutated EU amino acid positions.
  • the L234A/L235A mutant can be referred to as LALA. It is noted that the order in which substitutions are provided is arbitrary.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises variable regions having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises CDRs comprising the SEQ ID Nos disclosed herein with framework regions of the variable regions described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • an anti-CD1 17 antibody, or antigen binding fragment thereof has a certain dissociation rate.
  • an anti-CD1 17 antibody has, in certain embodiments, an off rate constant (Koff) for human CD1 17 and/or rhesus CD1 17 of 1 x 10 "2 to 1 x 10 "3 , 1 x 10 "3 to 1 x 10 ⁇ 4 , 1 x 10 ⁇ 5 to 1 x 10 ⁇ 6 , 1 x 10 "6 to 1 x 10 "7 or 1 x 10 "7 to 1 x 10 ⁇ 8 , as measured by bio-layer interferometry (BLI).
  • Koff off rate constant
  • the antibody or antigen-binding fragment thereof binds CD1 17 (e.g., human CD1 17 and/or rhesus CD1 17) with a K D of about 100 nM or less, about 90nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nM or less as determined by a Bio-Layer Interferometry (BLI) assay.
  • BBI Bio-Layer Interferometry
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
  • isolated nucleic acid encoding an anti-CD1 17 antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1 ) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of making an anti-CLL-1 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed.,
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A);
  • human lung cells W138; human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises variable regions having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises CDRs comprising the SEQ ID Nos disclosed herein with framework regions of the variable regions described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region having an amino acid sequence that is disclosed herein.
  • the anti-CD1 17 antibody, or antigen binding fragment thereof comprises a light chain variable region and a light chain constant region having an amino acid sequence that is disclosed herein.
  • the anti- CD1 17 antibody, or antigen binding fragment thereof comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region having an amino acid sequence that is disclosed herein.
  • novel anti-CD1 17 antibodies that may be used, for example, in conditioning methods for stem cell transplantation.
  • other anti- CD1 17 antibodies e.g., neutral antibodies, can be identified.
  • Methods for high throughput screening of antibody, or antibody fragment libraries for molecules capable of binding CD1 17 can be used to identify and affinity mature antibodies useful for treating cancers, autoimmune diseases, and conditioning a patient (e.g., a human patient) in need of hematopoietic stem cell therapy as described herein.
  • Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others.
  • phage display to isolate ligands that bind biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev.
  • Randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind cell surface antigens as described in Kay, Perspect. Drug Discovery Des. 2:251 -268, 1995 and Kay et al., Mol. Divers. 1 :139-140, 1996, the disclosures of each of which are incorporated herein by reference as they pertain to the discovery of antigen-binding molecules.
  • Proteins such as multimeric proteins have been successfully phage-displayed as functional molecules (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as they pertain to the use of in vitro display techniques for the discovery of antigen-binding molecules).
  • functional antibody fragments such as Fab and scFv fragments, have been expressed in in vitro display formats (see, for example, McCafferty et al., Nature 348:552- 554, 1990; Barbas et al., Proc. Natl. Acad. Sci.
  • computational modeling techniques can be used to design and identify antibodies, and antibody fragments, in silico that bind CD1 17 (e.g., GNNK+ CD1 1 7).
  • CD1 17 e.g., GNNK+ CD1 1 7
  • computational modeling techniques one of skill in the art can screen libraries of antibodies, and antibody fragments, in silico for molecules capable of binding specific epitopes, such as extracellular epitopes of this antigen.
  • the antibodies, and antigen-binding fragments thereof, identified by these computational techniques can be used in conjunction with the therapeutic methods described herein, such as the cancer and autoimmune disease treatment methods described herein and the patient conditioning procedures described herein.
  • Additional techniques can be used to identify antibodies, and antigen-binding fragments thereof, that bind CD1 17 (e.g., GNNK+ CD1 1 7) on the surface of a cell (e.g., a cancer cell, autoimmune cell, or hematopoietic stem cell) and that are internalized by the cell, for instance, by receptor-mediated endocytosis.
  • a cell e.g., a cancer cell, autoimmune cell, or hematopoietic stem cell
  • the in vitro display techniques described above can be adapted to screen for antibodies, and antigen-binding fragments thereof, that bind CD1 17 (e.g., GNNK+ CD1 17) on the surface of a cancer cell, autoimmune cell, or hematopoietic stem cell and that are subsequently internalized.
  • Phage display represents one such technique that can be used in conjunction with this screening paradigm.
  • CD1 1 7 e.g., GNNK+ CD1 17
  • GNNK+ CD1 17 bind CD1 1 7
  • autoimmune cells e.g., IL-12
  • hematopoietic stem cells e.g., IL-12 1 7
  • one of skill in the art can adapt the phage display techniques described, for example, in Williams et al., Leukemia 1 9:1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety.
  • recombinant phage libraries can be produced that encode antibodies, antibody fragments, such as scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domains, among others, or ligands that contain randomized amino acid cassettes (e.g., in one or more, or all, of the CDRs or equivalent regions thereof or an antibody or antibody fragment).
  • the framework regions, hinge, Fc domain, and other regions of the antibodies or antibody fragments may be designed such that they are non-immunogenic in humans, for instance, by virtue of having human germline antibody sequences or sequences that exhibit only minor variations relative to human germline antibodies.
  • phage libraries containing randomized antibodies, or antibody fragments, covalently bound to the phage particles can be incubated with CD1 1 7 (e.g., GNNK+ CD1 17) antigen, for instance, by first incubating the phage library with blocking agents (such as, for instance, milk protein, bovine serum albumin, and/or IgG so as to remove phage encoding antibodies, or fragments thereof, that exhibit nonspecific protein binding and phage that encode antibodies or fragments thereof that bind Fc domains, and then incubating the phage library with a population of hematopoietic stem cells.
  • blocking agents such as, for instance, milk protein, bovine serum albumin, and/or IgG
  • the phage library can be incubated with the target cells, such as cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to allow CD1 1 7-specific antibodies, or antigen- binding fragments thereof, (e.g., GNNK+ CD1 17-specific antibodies, or antigen-binding fragments thereof) to bind cell-surface CD1 17 (e.g., sell-surface GNNK+ CD1 17) antigen and to the target cells, such as cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to allow CD1 1 7-specific antibodies, or antigen- binding fragments thereof, (e.g., GNNK+ CD1 17-specific antibodies, or antigen-binding fragments thereof) to bind cell-surface CD1 17 (e.g., sell-surface GNNK+ CD1 17) antigen and to
  • hematopoietic stem cells e.g., from 30 minutes to 6 hours at 4° C, such as 1 hour at 4° C.
  • Phage containing antibodies, or fragments thereof, that do not exhibit sufficient affinity for one or more of these antigens so as to permit binding to, and internalization by, cancer cells, autoimmune cells, or hematopoietic stem cells can subsequently be removed by washing the cells, for instance, with cold (4° C) 0.1 M glycine buffer at pH 2.8.
  • Phage bound to antibodies, or fragments thereof, that have been internalized by the cancer cells, autoimmune cells, or hematopoietic stem cells can be identified, for instance, by lysing the cells and recovering internalized phage from the cell culture medium.
  • the phage can then be amplified in bacterial cells, for example, by incubating bacterial cells with recovered phage in 2xYT medium using methods known in the art.
  • Phage recovered from this medium can then be characterized, for instance, by determining the nucleic acid sequence of the gene(s) encoding the antibodies, or fragments thereof, inserted within the phage genome.
  • the encoded antibodies, or fragments thereof can subsequently be prepared de novo by chemical synthesis (for instance, of antibody fragments, such as scFv fragments) or by recombinant expression (for instance, of full-length antibodies).
  • Phage display libraries can be created by making a designed series of mutations or variations within a coding sequence for the CDRs of an antibody or the analogous regions of an antibody-like scaffold (e.g., the BC, CD, and DE loops of 10 Fn3 domains).
  • the template antibody-encoding sequence into which these mutations are introduced may be, for example, a naive human germline sequence. These mutations can be performed using standard mutagenesis techniques known in the art. Each mutant sequence thus encodes an antibody corresponding to the template save for one or more amino acid variations.
  • Retroviral and phage display vectors can be engineered using standard vector construction techniques known in the art. P3 phage display vectors along with compatible protein expression vectors can be used to generate phage display vectors for antibody diversification.
  • the mutated DNA provides sequence diversity, and each transformant phage displays one variant of the initial template amino acid sequence encoded by the DNA, leading to a phage population (library) displaying a vast number of different but structurally related amino acid sequences. Due to the well-defined structure of antibody hypervariable regions, the amino acid variations introduced in a phage display screen are expected to alter the binding properties of the binding peptide or domain without significantly altering its overall molecular structure.
  • a phage library may be contacted with and allowed to bind one of the foregoing antigens or an epitope thereof.
  • Phage bearing a CD1 17-binding moiety can form a complex with the target on the solid support, whereas non-binding phage remain in solution and can be washed away with excess buffer.
  • Bound phage can then liberated from the target by changing the buffer to an extreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, or other known means.
  • the recovered phage can then be amplified through infection of bacterial cells, and the screening process can be repeated with the new pool that is now depleted in non-binding antibodies and enriched for antibodies that bind CD1 17 (e.g., GNNK+ CD1 17).
  • the recovery of even a few binding phage is sufficient to amplify the phage for a subsequent iteration of screening.
  • the gene sequences encoding the antibodies or antigen-binding fragments thereof derived from selected phage clones in the binding pool are determined by conventional methods, thus revealing the peptide sequence that imparts binding affinity of the phage to the target.
  • sequence diversity of the population diminishes with each round of selection until desirable peptide-binding antibodies remain.
  • the sequences may converge on a small number of related antibodies or antigen-binding fragments thereof.
  • An increase in the number of phage recovered at each round of selection is an indication that convergence of the library has occurred in a screen.
  • Another method for identifying anti-CD1 17 antibodies includes using humanizing non- human antibodies that bind CD1 17 (e.g., GNNK+ CD1 17), for instance, according to the following procedure.
  • Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the "VBASE" human germline sequence database; Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242, 1991 ; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al.. Eur. J. Immunol. 24:827- 836, 1994, the disclosures of each of which are incorporated herein by reference as they pertain to consensus human antibody heavy chain and light chain
  • variable domain framework residues and CDRs of a consensus antibody sequence e.g., by sequence alignment.
  • This CDR exchange can be performed using gene editing techniques described herein or known in the art.
  • consensus human antibody that may be used in the preparation of a humanized antibody comprises a heavy chain variable domain set forth in SEQ ID NO: 7:
  • EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADS VKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain set forth in SEQ ID NO: 8: DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 8), identified in US Patent No. 6,054,297 (Genentech), the disclosure of which is incorporated herein by reference as it pertains to human antibody consensus sequences. The CDRs in the above sequences are shown in bold.
  • variable region CDRs binds CD1 17 (e.g., GNNK+ CD1 17).
  • GNNK+ CD1 17 a non-human antibody that binds CD1 17
  • the affinity of the antibody for the hematopoietic stem cell antigen is determined primarily by the CDR sequences, the resulting humanized antibody is
  • an antibody for a target antigen that is about the same as that of the non-human antibody from which the humanized antibody was derived.
  • Methods of determining the affinity of an antibody for a target antigen include, for instance, ELISA-based techniques described herein and known in the art, as well as surface plasmon resonance,
  • the internalizing capacity of the anti-CD1 17 antibodies, or fragments thereof can be assessed, for instance, using radionuclide internalization assays known in the art.
  • antibodies, or fragments thereof, identified using in vitro display techniques described herein or known in the art can be functionalized by incorporation of a radioactive isotope, such as 18 F, 75 Br, 77 Br, 122 l, 123 l, 124 l, 125 l, 129 l, 131 l, 211 At, 67 Ga, 111 ln, "Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac.
  • a radioactive isotope such as 18 F, 75 Br, 77 Br, 122 l, 123 l, 124 l, 125 l, 129 l, 131 l, 211 At, 67
  • radioactive halogens such as 18 F, 75 Br, 77 Br, 122 l, 123 l, 124 l, 125 l, 129 l, 131 1, 211 At, can be incorporated into antibodies, or fragments thereof, using beads, such as polystyrene beads, containing electrophilic halogen reagents (e.g., lodination Beads, Thermo Fisher Scientific, Inc., Cambridge, MA).
  • Radiolabeled antibodies, or fragments thereof can be incubated with cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to permit internalization (e.g., from 30 minutes to 6 hours at 4° C, such as 1 hour at 4° C).
  • the cells can then be washed to remove non-internalized antibodies, or fragments thereof, (e.g., using cold (4° C) 0.1 M glycine buffer at pH 2.8).
  • Internalized antibodies, or fragments thereof can be identified by detecting the emitted radiation (e.g., ⁇ -radiation) of the resulting cancer cells, autoimmune cells, or hematopoietic stem cells in comparison with the emitted radiation (e.g., ⁇ -radiation) of the recovered wash buffer.
  • hematopoietic stem cell transplant therapy can be administered to a subject in need of treatment so as to populate or re-populate one or more blood cell types.
  • Hematopoietic stem cells generally exhibit multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes,
  • Hematopoietic stem cells are additionally capable of self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and also feature the capacity to be reintroduced into a transplant recipient whereupon they home to the
  • Hematopoietic stem cells can thus be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo, thereby treating the pathology associated with the defect or depletion in the endogenous blood cell population.
  • the compositions and methods described herein can thus be used to treat a non-malignant hemoglobinopathy (e.g., a hemoglobinopathy selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and
  • compositions and methods described herein can be used to treat an immunodeficiency, such as a congenital immunodeficiency.
  • compositions and methods described herein can be used to treat an acquired immunodeficiency (e.g., an acquired immunodeficiency selected from the group consisting of HIV and AIDS).
  • the compositions and methods described herein can be used to treat a metabolic disorder (e.g., a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy).
  • compositions and methods described herein can be used to treat a malignancy or proliferative disorder, such as a hematologic cancer, myeloproliferative disease.
  • a malignancy or proliferative disorder such as a hematologic cancer, myeloproliferative disease.
  • the compositions and methods described herein may be administered to a patient so as to deplete a population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation therapy, in which case the transplanted cells can home to a niche created by the endogenous cell depletion step and establish productive hematopoiesis. This, in turn, can re-constitute a population of cells depleted during cancer cell eradication, such as during systemic chemotherapy.
  • Exemplary hematological cancers that can be treated using the compositions and methods described herein include, without limitation, acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, as well as other cancerous conditions, including neuroblastoma.
  • Additional diseases that can be treated with the compositions and methods described herein include, without limitation, adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.
  • compositions and methods described herein may be used to induce solid organ transplant tolerance.
  • the compositions and methods described herein may be used to deplete or ablate a population of cells from a target tissue (e.g., to deplete hematopoietic stem cells from the bone marrow stem cell niche).
  • a population of stem or progenitor cells from an organ donor e.g., hematopoietic stem cells from the organ donor
  • a temporary or stable mixed chimerism may be achieved, thereby enabling long-term transplant organ tolerance without the need for further immunosuppressive agents.
  • compositions and methods described herein may be used to induce transplant tolerance in a solid organ transplant recipient (e.g., a kidney transplant, lung transplant, liver transplant, and heart transplant, among others).
  • a solid organ transplant recipient e.g., a kidney transplant, lung transplant, liver transplant, and heart transplant, among others.
  • the compositions and methods described herein are well-suited for use in connection the induction of solid organ transplant tolerance, for instance, because a low percentage temporary or stable donor engraftment is sufficient to induce long-term tolerance of the transplanted organ.
  • compositions and methods described herein can be used to treat cancers directly, such as cancers characterized by cells that are CD1 17+.
  • cancers characterized by cells that are CD1 17+.
  • the compositions and methods described herein can be used to treat leukemia, particularly in patients that exhibit CD1 17+ leukemic cells.
  • the compositions and methods described herein can be used to treat various cancers directly.
  • Exemplary cancers that may be treated in this fashion include hematological cancers, such as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma,
  • hematological cancers such as acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma
  • compositions and methods described herein can be used to treat autoimmune disorders.
  • an antibody, or antigen-binding fragment thereof can be administered to a subject, such as a human patient suffering from an autoimmune disorder, so as to kill a CD1 17+ immune cell.
  • the CD1 17+ immune cell may be an autoreactive lymphocyte, such as a T-cell that expresses a T-cell receptor that specifically binds, and mounts an immune response against, a self-antigen.
  • the compositions and methods described herein can be used to treat autoimmune pathologies, such as those described below.
  • compositions and methods described herein can be used to treat an autoimmune disease by depleting a population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation therapy, in which case the transplanted cells can home to a niche created by the endogenous cell depletion step and establish productive hematopoiesis. This, in turn, can re-constitute a population of cells depleted during autoimmune cell eradication.
  • Autoimmune diseases that can be treated using the compositions and methods described herein include, without limitation, psoriasis, psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes), rheumatoid arthritis (RA), human systemic lupus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitisis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn
  • Antibodies, or antigen-binding fragments thereof, described herein can be administered to a patient (e.g., a human patient suffering from cancer, an autoimmune disease, or in need of hematopoietic stem cell transplant therapy) in a variety of dosage forms.
  • a patient e.g., a human patient suffering from cancer, an autoimmune disease, or in need of hematopoietic stem cell transplant therapy
  • antibodies, or antigen-binding fragments thereof, described herein can be administered to a patient suffering from cancer, an autoimmune disease, or in need of hematopoietic stem cell transplant therapy in the form of an aqueous solution, such as an aqueous solution containing one or more
  • compositions and methods described herein include viscosity-modifying agents.
  • aqueous solution may be sterilized using techniques known in the art.
  • compositions comprising anti-CD1 17 antibodies as described herein are prepared by mixing such antibodies with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable carriers Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid and methionine
  • preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl
  • polypeptides such as serum albumin, gelatin, or immunoglobulins
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as polyethylene glycol (PEG).
  • the antibodies, or antigen-binding fragments, described herein may be administered by a variety of routes, such as orally, transdermal ⁇ , subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally.
  • routes such as orally, transdermal ⁇ , subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally.
  • the most suitable route for administration in any given case will depend on the particular antibody, or antigen-binding fragment, administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate.
  • the effective dose of an antibody, or antigen-binding fragment thereof, described herein can range, for example from about 0.001 to about 100 mg/kg of body weight per single (e.g., bolus) administration, multiple administrations, or continuous administration, or to achieve an optimal serum concentration (e.g., a serum concentration of 0.0001 -5000 g/mL) of the antibody, or antigen-binding fragment thereof.
  • the dose may be administered one or more times (e.g., 2-10 times) per day, week, or month to a subject (e.g., a human) suffering from cancer, an autoimmune disease, or undergoing conditioning therapy in preparation for receipt of a hematopoietic stem cell transplant. In the case of a conditioning procedure prior to hematopoietic stem cell
  • the antibody, or antigen-binding fragment thereof can be administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) or more prior to administration of the exogenous hematopoietic stem cell transplant.
  • 1 hour to 1 week e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours,
  • a physician of skill in the art can administer to a human patient in need of hematopoietic stem cell transplant therapy an antibody or antigen- binding fragment thereof capable of binding an antigen expressed by hematopoietic stem cells, such as an antibody or antigen-biding fragment thereof that binds CD1 17 (for example, an antibody or antigen-binding fragment thereof that binds GNNK+ CD1 17).
  • an antibody or antigen-binding fragment thereof capable of binding an antigen expressed by hematopoietic stem cells, such as an antibody or antigen-biding fragment thereof that binds CD1 17 (for example, an antibody or antigen-binding fragment thereof that binds GNNK+ CD1 17).
  • the antibody, or antigen-binding fragment thereof can subsequently be administered to the patient, for example, by intravenous administration, prior to transplantation of exogenous hematopoietic stem cells (such as autologous, syngeneic, or allogeneic hematopoietic stem cells) to the patient.
  • exogenous hematopoietic stem cells such as autologous, syngeneic, or allogeneic hematopoietic stem cells
  • the anti-CD1 17 (e.g., anti-GNNK+ CD1 17) antibody, or antigen-binding fragment thereof, can be administered in an amount sufficient to reduce the quantity of endogenous hematopoietic stem cells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior to hematopoietic stem cell transplant therapy.
  • the reduction in hematopoietic stem cell count can be monitored using conventional techniques known in the art, such as by FACS analysis of cells expressing characteristic hematopoietic stem cell surface antigens in a blood sample withdrawn from the patient at varying intervals during conditioning therapy.
  • a physician of skill in the art can withdraw a blood sample from the patient at various time points during conditioning therapy and determine the extent of endogenous hematopoietic stem cell reduction by conducting a FACS analysis to elucidate the relative concentrations of hematopoietic stem cells in the sample using antibodies that bind to hematopoietic stem cell marker antigens.
  • the physician may conclude the conditioning therapy, and may begin preparing the patient for hematopoietic stem cell transplant therapy.
  • the anti-CD1 17 (e.g., anti-GNNK+ CD1 17) antibody, or antigen-binding fragment thereof can be administered to the patient in an aqueous solution containing one or more pharmaceutically acceptable excipients, such as a viscosity-modifying agent.
  • the aqueous solution may be sterilized using techniques described herein or known in the art.
  • the antibody, or antigen-binding fragment thereof can be administered to the patient at a dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior to administration of a hematopoietic stem cell graft to the patient.
  • the antibody or antigen-binding fragment thereof can be administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) or more prior to administration of the exogenous hematopoietic stem cell transplant.
  • 1 hour to 1 week e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23
  • the patient may then receive an infusion (e.g., an intravenous infusion) of exogenous hematopoietic stem cells, such as from the same physician that performed the conditioning therapy or from a different physician.
  • an infusion e.g., an intravenous infusion
  • the physician may administer the patient an infusion of autologous, syngeneic, or allogeneic hematopoietic stem cells, for instance, at a dosage of from 1 x 10 3 to 1 x 10 9 hematopoietic stem cells/kg.
  • the physician may monitor the engraftment of the hematopoietic stem cell transplant, for example, by withdrawing a blood sample from the patient and determining the increase in concentration of hematopoietic stem cells or cells of the hematopoietic lineage (such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T- lymphocytes, and B-lymphocytes) following administration of the transplant.
  • hematopoietic stem cells or cells of the hematopoietic lineage such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia
  • This analysis may be conducted, for example, from 1 hour to 6 months, or more, following hematopoietic stem cell transplant therapy (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more).
  • hematopoietic stem cell transplant therapy e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours,
  • a finding that the concentration of hematopoietic stem cells or cells of the hematopoietic lineage has increased (e.g., by 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) following the transplant therapy relative to the concentration of the corresponding cell type prior to transplant therapy provides one indication that treatment with the anti-CD1 17 (e.g., anti-GNNK+ CD1 17) antibody, or antigen- binding fragment thereof, has successfully promoted engraftment of the transplanted
  • the anti-CD1 17 e.g., anti-GNNK+ CD1 17
  • anti-CD1 17 antibodies provided herein may be used in a variety of non-therapeutic methods, such as in affinity purification methods or diagnostic assays.
  • an anti-CD1 17 antibody herein is used as an affinity purification agent, e.g., to purify CD1 17.
  • the antibodies are immobilized on a solid phase such as Sephadex resin or filter paper, using methods well known in the art.
  • an anti-CD1 17 antibody herein may also be useful in diagnostic assays for CD1 17 protein, e.g., detecting its expression in specific cells, tissues, or serum.
  • the antibody typically will be labeled with a detectable moiety.
  • a detectable moiety Numerous labels are available which can be preferably grouped into the following categories:
  • Radioisotopes such as 35S, 14C, 1251, 3H, and 131 1.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed., Wiley-lnterscience, New York, N.Y., Pubs., (1991 ) for example and radioactivity can be measured using scintillation counting.
  • Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • the enzyme may catalyze a color change in a substrate, which can be measured
  • the enzyme may alter the fluorescence or
  • chemiluminescence of the substrate involves techniques for quantifying a change in fluorescence.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.
  • luciferin 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • glucose oxidase galactose oxidase
  • enzyme-substrate combinations include, for example:
  • HRPO Horseradish peroxidase
  • HPO horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'- tetramethyl benzidine hydrochloride
  • alkaline phosphatase AP
  • para-Nitrophenyl phosphate as chromogenic substrate
  • ⁇ -D-galactosidase ⁇ -D-Gal
  • a chromogenic substrate e.g. p-nitrophenyl- ⁇ - ⁇ - galactosidase
  • fluorogenic substrate 4-methylumbelliferyl ⁇ -D-galactosidase
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody).
  • a small hapten e.g. digoxin
  • an anti-hapten antibody e.g. anti-digoxin antibody
  • the anti-CD1 17 antibody need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the anti-CD1 17 antibody.
  • An anti-CD1 17 antibody herein may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
  • ком ⁇ онентs rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody.
  • the amount of CD1 17 protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies preferably are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • the antibodies may also be used for in vivo diagnostic assays.
  • CD1 17 antibody may be labelled with a radionuclide (such as 1 1 1 1n, 99Tc, 14C, 131 1, 125I, 3H, 32P or 35S) so that the tumor can be localized using immunoscintigraphy.
  • a radionuclide such as 1 1 1 1n, 99Tc, 14C, 131 1, 125I, 3H, 32P or 35S
  • kits i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g. a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g. a substrate precursor which provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (e.g. a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is the anti-CD1 17 antibody.
  • the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringers solution and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a yeast display library that displays antibodies (either natural or synthetic) was screened for binding to the ectodomain of human CD1 17.
  • Yeast cells that encoded antibodies that bound to human CD1 17 were selected.
  • Nucleic acid sequences representing the antibodies from the selected yeast cells were isolated according to techniques known in the art.
  • the screen was performed to identify antagonist anti-CD1 17 antibodies or neutral antibodies.
  • Neutral antibodies provide the benefit of being inert on target.
  • Antagonistic antibodies e.g., an antagonistic anti-CD1 17 antibody, prevent the association of CD1 17 with SCF (stem cell factor).
  • SCF stem cell factor
  • a neutral antibody may be therapeutically more safe, because, for example, antagonistic activity may negatively impact a graft by delaying engraftment via engaging with a donor CD1 17-bearing cells. A neutral antibody would circumvent this issue.
  • the yeast display technique described below was utilized to identify such neutral antibodies in addition to antagonist antibodies.
  • CD1 17 ectodomain was pre-complexed with the natural ligand Stem Cell Factor (SCF) and only antibodies capable of binding CD1 17 ectodomain in this complex were selected. Based on this selection method, isolated antibodies would not prevent the binding of SCF to CD1 17, classifying them as neutral.
  • SCF Stem Cell Factor
  • antibodies were also selected for their ability to internalize in a CD1 17 expressing cell, such as a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • Approximately 69 antibodies were initially obtained in the screening of a yeast library expressing recombinant fully human IgGs. From the 69 antibodies, 6 human IgG antibodies were selected for affinity maturation for further improvement by way of diversifying sequences in CDR1 and CDR2 in the heavy chain and selecting for improved affinity according to methods known in the art. 22 human IgG antibodies with improved affinity and variant sequences were subsequently identified following affinity maturation. From the overall screening process, 10 human IgG antibodies were selected based on desired binding properties, including 7 following affinity maturation and 3 antibodies identified prior to affinity maturation.
  • anti-CD1 17 antibodies were expressed and the resulting antibodies were further screened to identify anti-CD1 17 antibodies having desired structure and/or functional activity (e.g., the screen selected for antagonistic (ligand blocking) antibodies with cell internalization properties or for neutral antibodies having cell internalization properties).
  • desired structure and/or functional activity e.g., the screen selected for antagonistic (ligand blocking) antibodies with cell internalization properties or for neutral antibodies having cell internalization properties.
  • methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, Boder E.T. and Wittrup K.D., Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol, 328:430-44 (2000) and Boder E.T. and Wittrup K.D., Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol.
  • the amino acid sequences of the variable regions and CDRs of the heavy and light chains of the 10 selected antibodies are provided in Table 10.
  • the 10 antibodies include the following: antibody 54 (Ab54 having heavy chain (HC)-54 and light chain (LC)-54), Antibody 55 (Ab55 having HC-55 and LC-55), Antibody 56 (Ab56 having HC-56 and LC-56), Antibody 57 (Ab57 having HC- 57 and LC-57), Antibody 58 (Ab58 having HC-58 and LC-58), Antibody 61 (Ab61 having HC-61 and LC-61 ), Antibody 66 (Ab66 having HC-66 and LC-66), Antibody 67 (Ab67 having HC-67 and LC-67), Antibody 68 (Ab68 having HC-68 and LC-68), and Antibody 69 (Ab69 having HC-69 and LC-69).
  • Example 2 In vitro binding analysis of anti-CD117 antibodies
  • the apparent monovalent affinity (K D ), apparent association rate (K 0N ), and apparent dissociation rate (K 0F F) were determined by local full fitting with a 1 :1 binding model as calculated by ForteBio data analysis software version 10 of each IgG to purified human CD1 17 ectodomain (R&D Systems #332-SR) or rhesus CD1 17 ectodomain are shown in Table 1 .
  • each of the selected antibodies was able to cross react with rhesus CD1 17 and human CD1 17, specifically, the CD1 17 ectodomain.
  • the selected antibodies were also able to bind to both isoforms (1 and 2) of human CD1 17.
  • Table 1 Monovalent affinity (K D ), apparent association rate (K 0N ), and apparent dissociation rate (KQFF) of the indicated IgG to human CD1 1 7 ectodomain or rhesus CD1 1 7 ectodomain
  • Antibody 67 was identified in the above screen described in Example 1 in particular for being a neutral antibody.
  • the heavy chain and light chain variable regions of Ab67 are described below in Table 2.
  • VH variable region amino acid sequence of Ab67 is described in SEQ ID NO: 1
  • VH CDR amino acid sequences of Ab67 are as follows: FTFSDADMD (VH CDR1 ; SEQ ID NO: 1 1 ); RTRNKAGSYTTEYAASVKG (VH CDR2; SEQ ID NO: 12); and
  • AREPKYWIDFDL VH CDR3; SEQ ID NO: 13
  • VL variable region amino acid sequence of Ab67 is provided below as SEQ ID NO 10.
  • VL CDR amino acid sequences of HC-67/LC-67 are underlined below and are as follows: RASQSISSYLN (VL CDR1 ; SEQ ID NO: 14); AASSLQS (VL CDR2; SEQ ID NO: 15); and QQSYIAPYT (VL CDR3; SEQ ID NO: 16).
  • the binding characteristics of Ab67 to bind human CD1 17 were studied using a standard biolayer interferometry binding assay.
  • Antibody 67 (an lgG1 ) binding studies were performed at 25 degrees Celsius in 1 x PBS supplemented with 0.1 % w/v bovine serum albumin with a Pall ForteBio Octet Red96 using biolayer interferometry (BLI).
  • the indicated purified human antibody was immobilized onto anti-human Fc biosensors (AHC; Pall ForteBio 18-5063) and incubated with 33.3 nM and 1 1 nM CD1 17 ectodomain (R&D Systems #332-SR)
  • the resulting binding intervals which represented the association and dissociation curves, were depicted in Fig. 1 .
  • the apparent monovalent affinity (KD), apparent association rate (kon), and apparent dissociation rate (kdis) were determined by local full fitting with a 1 :1 binding model as calculated by Fortebio data analysis software version 10 of the indicated purified IgG (i.e., HC-67/LC-67) to purified human CD1 17 ectodomain (R&D Systems #332-SR) as shown in Table 3.
  • human CD34+ bone marrow cells were cultured for 5 days in the presence of stem-cell factor and the control antibody 3100 mAb (3100 corresponds to anti-CD1 17 antibody CK6; a known anti-CD1 17 antagonist antibody), the HC-67/LC-67 (Ab67) IgG (lgG1 ) or the isotype control (i.e., hlgG1 ). Full length IgG antibodies were tested. Live cell counts were determined for all cells or CD34+ CD90+ gated cells by flow cytometry.
  • HC-67/LC-67 is a neutral non-antagonist antibody that does not prohibit the SCF-dependent proliferation of primary human CD34+ bone marrow cells in culture, as the activity of Ab67 was similar to the isotype matched negative control. Note that the protocol for the SCF cell proliferation assay used in this example is described in Example 8.
  • Antibody 55 was identified in the above yeast screen for having properties corresponding to a therapeutic human anti-CD1 17 antibody, particularly antagonistic
  • a 1 1 1 1 1 GCAACTTACTACTGTCAG (SEQ ID NO: 20)
  • VH variable region amino acid sequence of Ab55 is described in SEQ ID NO: 19.
  • VH CDR amino acid sequences of Ab55 are as follows: GTFRIYAIS (VH CDR1 ; SEQ ID NO: 21 ); GIIPDFGVANYAQKFQG (VH CDR2; SEQ ID NO: 22); and ARGGLDTDEFDL (VH CDR3; SEQ ID NO: 23).
  • VL variable region amino acid sequence of Ab55 is provided below as SEQ ID NO 20.
  • VL CDR amino acid sequences of HC-55/LC-55 are underlined below and are as follows: RASQSINSYLN (VL CDR1 ; SEQ ID NO: 24); AASSLQS (VL CDR2; SEQ ID NO: 25); and QQGVSDIT (VL CDR3; SEQ ID NO: 26).
  • RASQSINSYLN VL CDR1 ; SEQ ID NO: 24
  • AASSLQS VL CDR2; SEQ ID NO: 25
  • QQGVSDIT VL CDR3; SEQ ID NO: 26.
  • the apparent monovalent affinity (KD), apparent association rate (kon), and apparent dissociation rate (kdis) are determined by local full fitting with a 1 :1 binding model as calculated by ForteBio data analysis software version 10 of the indicated purified IgG (i.e., HC-55/LC-55) to purified human CD1 17 ectodomain (R&D Systems #332-SR) are shown in Table 5.
  • the results demonstrate an Ab55 IgG (i.e., the HC- 55/LC-55 IgG) binds with high affinity to the purified human CD1 17 ectodomain and was also characterized as having a slow kdis(1/s) as determined by standard BLI.
  • human CD34+ bone marrow cells were cultured for 5 days in the presence of stem-cell factor, the 3100 mAb, the HC-55/LC-55 IgG (Ab55 lgG1 ) or the control (i.e., hlgG1 ). Live cell counts were determined for all cells or CD34+ CD90+ gated cells by flow cytometry.
  • Figs. 4A and 4B demonstrate the dose dependent effect of the 3100 mAb (antagonist CD1 17 antibody CK6) and the HC-55/LC-55 IgG in the SCF-dependent proliferation of human CD34+ cells as measured by flow cytometry.
  • This result demonstrates that HC-55/LC-55 is an antagonist antibody that prohibits the SCF-dependent proliferation of primary human CD34+ bone marrow cells in culture similar to the 3100 mAb.
  • Example 8 Analysis of anti-CD117 antibodies using an in vitro SCF dependent cell proliferation assay
  • SCF stem cell factor
  • CK6 was used as a positive control, as it is known to have antagonist activity (see U.S. Patent No. 8,552,157), and an isotype non-CD1 17 binding antibody was used as a negative control.
  • human CD34+ bone marrow cells were cultured for 5 days in the presence of SCF and the indicated antibody. Live cell counts were determined for all cells or for CD34+ CD90+ gated cells by flow cytometry.
  • Figs. 6A and 6B live cells
  • Figs. 7A and 7B CD34+ CD90+ cells
  • antibodies Ab54, Ab55, Ab56, and Ab57 are CD1 17 antagonistic as they were able to kill both live and CD34+ CD90 cells in an SCF dependent cell proliferation assay and prevent SCF- dependent proliferation.
  • Ab58, Ab61 , Ab66, Ab67, Ab68, and Ab69 were determined to be neutral antibodies that do not inhibit the SCF-dependent proliferation of human CD34+ bone marrow cells in culture.
  • Ab67 and Ab55 were further assessed in a cross-blocking assay in the presence of CK6 (anti-CD1 17 antagonist antibody) or human SCF (the ligand to CD1 17).
  • the cross-blocking assay was performed at 25 degrees Celsius in 1 x PBS supplemented with 0.1 % w/v bovine serum albumin with a Pall ForteBio Octet Red96 using biolayer interferometry (BLI).
  • the indicated purified human antibody was immobilized onto anti-human Fc biosensors (AHC; Pall ForteBio 18- 5063) and incubated with 100 nM CD1 17 ectodomain (R&D Systems #332-SR).
  • Figs. 9A and 9B provide data from a second cross-blocking assay testing whether Ab67 could crossblock either human or mouse SCF from binding to huCD1 17.
  • CD1 17 ectodomain 25 nM of biotinylated recombinant CD1 17 ectodomain (R&D Systems #332-SR) was immobilized onto streptavidin biosensors (SA; Pall ForteBio 18-5019) and incubated with 100 nM of Ab67.
  • SA streptavidin biosensors
  • the CD1 17 ectodomain in complex with Ab67 was incubated with human (ThermoFisher Scientific PHC21 13) or mouse (R&D Systems #455-MC) stem cell factor.
  • Fig. 9B Ab67 was not able to inhibit binding of either huSCF or mSCF to huCD1 17, thus further providing evidence of the neutral characteristic of Ab67.
  • the internalizing capacity of Ab67 versus an anti-CD1 17 antagonistic antibody was assessed in an in vitro antibody internalization assay. This assay was performed by incubating human bone marrow CD34+ cells with a saturating concentration of either a neutral, antagonistic, or control hlgG1 antibody over 24 hours. At the conclusion of the time course, a fluorophore- labeled anti-lgG molecule was used to assess remaining surface hlgG1 by flow cytometry. The percent of surface IgG was examined over time (Fig. 10B). Normalized CD1 17 surface expression (Fig. 10A) was calculated with a control condition incubated at 4 degrees C where internalization would not be expected (not shown).
  • the percent of CD1 17 on the surface of the cells was lower in the presence of neutral antibody Ab67 (lgG1 ) as compared to that observed following treatment with an antagonist anti-CD1 17 antibody or an isotype-matched hlgG1 .
  • CD1 17 internalization of CD1 17 was similar to that of SCF. Further, as described in Fig. 10B, the percent of neutral antibody Ab67 on the surface of the cells decreased over time relative to control antagonist or hlgG1 antibodies. This also suggests that the neutral anti-CD1 17 antibody, Ab67, internalized more rapidly than antagonist antibodies.
  • hydrophobicity of each of Ab55 and AB67 was determined using hydrophobic interaction chromatography (HIC).
  • Anti-CD1 17 antibody CK6 was used as a control for comparison.
  • Antibodies Ab55 and Ab67 were evaluated after incubation at 25 or 50 degrees Celsius for 7 days or 15 days by hydrophobic interaction chromatography (HIC; Figs. 1 1 A-1 1 F). Briefly, 50 micrograms of the indicated antibody were injected onto a Tosoh TSKgel Phenyl-5PW 7.5 mm ID x 7.5 cm 10-micron column (Catalog # 07573) on a Waters ARC HPLC/UPLC system. For CK6 (Figs.
  • Capillary isoelectric focusing was performed on Ab55 and Ab67 antibodies to determine if sequence differentiation impacted the biophysical properties of the antibodies. Briefly, 10-40 micrograms of antibody were subjected to 7- and 15-days incubation at 25 or 50 degrees Celsius and analyzed through a capillary electrophoresis method using the Maurice instrument manufactured by Protein Simple according to standard manufacturer instruction. Antibody samples migrate to their electrically neutral pH. Acidic variants can be identified based on absorbance peaks detected below the isoelectric point relative to the total injected sample. The

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Abstract

L'invention concerne des compositions et des méthodes utiles pour la déplétion de cellules CD117+ et pour le traitement de diverses maladies hématopoïétiques, de troubles métaboliques, de cancers et de maladies auto-immunes, entre autres. L'invention concerne des anticorps, des fragments de ceux-ci de liaison à l'antigène, qui peuvent être appliqués à effectuer le traitement de ces affections, par exemple, par déplétion d'une population de cellules CD117+ chez un patient, tel qu'un être humain. Les compositions et les méthodes décrites dans la présente description peuvent être utilisées pour traiter un trouble directement, par exemple, par déplétion d'une population de cellules cancéreuses CD117+ ou de cellules auto-immunes. Les compositions et les méthodes décrites dans la présente description peuvent également être utilisées pour préparer un patient pour une thérapie par greffe de cellules souches hématopoïétiques et pour améliorer la prise de greffe de greffons de cellules souches hématopoïétiques par déplétion sélective de cellules souches hématopoïétiques endogènes avant l'intervention de greffe.
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WO2020219774A1 (fr) * 2019-04-24 2020-10-29 Magenta Therapeutics, Inc. Conjugués anticorps-médicaments d'antracycline et leurs utilisations
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EP3825330A1 (fr) * 2019-11-19 2021-05-26 International-Drug-Development-Biotech Anticorps anti-cd117 et leurs procédés d'utilisation
WO2021107566A1 (fr) * 2019-11-25 2021-06-03 주식회사 노벨티노빌리티 Anticorps dirigé contre c-kit et utilisation correspondante
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WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024163905A1 (fr) 2023-02-03 2024-08-08 Genzyme Corporation Nanoparticules lipidiques conjuguées à un anticorps spécifique de csh et utilisations associées
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WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024163905A1 (fr) 2023-02-03 2024-08-08 Genzyme Corporation Nanoparticules lipidiques conjuguées à un anticorps spécifique de csh et utilisations associées
WO2024249954A1 (fr) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Formulations et compositions de nanoparticules lipidiques

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