WO2017147247A1 - Anticorps anti-sas1b et ses méthodes d'utilisation - Google Patents

Anticorps anti-sas1b et ses méthodes d'utilisation Download PDF

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Publication number
WO2017147247A1
WO2017147247A1 PCT/US2017/019052 US2017019052W WO2017147247A1 WO 2017147247 A1 WO2017147247 A1 WO 2017147247A1 US 2017019052 W US2017019052 W US 2017019052W WO 2017147247 A1 WO2017147247 A1 WO 2017147247A1
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Prior art keywords
seq
antibody
sas1
antigen
cdr3
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PCT/US2017/019052
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English (en)
Inventor
Eusebio S. PIRES
Jagathpala Shetty
Brian Pollock
John C. Herr
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Pires Eusebio S
Jagathpala Shetty
Brian Pollock
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Application filed by Pires Eusebio S, Jagathpala Shetty, Brian Pollock filed Critical Pires Eusebio S
Publication of WO2017147247A1 publication Critical patent/WO2017147247A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96419—Metalloendopeptidases (3.4.24)

Definitions

  • One embodiment provides an isolated antibody or antigen-binding portion thereof comprising: (a) a VH CDR1 of SEQ ID NO: 1 , 7, 9, 14, 25, 31 , 37, 43, 48, or 54; a VH CDR2 of SEQ ID NO:2, 10, 15, 26, 32, 38, 44, 49, or 55; a VH CDR3 of SEQ ID NO:3, 8, 16, 27, 33, 39, 45, 50, 56, or GGL; a VL CDR1 of SEQ ID NO:4, 1 1 , 17, 28, 34, 40, 46, 51 , or 57; a VL CDR2 of SEQ ID NO:5, 12, 18, 29, 35, 41 , 47, 35, or 58; and a VL CDR3 of SEQ ID NO:6, 13, 19, 30, 36, 42, 53, or 59; or (b) a VH CDR1 of SEQ ID NO:20, a VH CDR2 of SEQ ID NO:21 , a VH CDR3 of SEQ ID NO:22, a
  • the antibody comprises: (a) a VH CDR1 of SEQ ID NO: 1 , a VH CDR2 of SEQ ID NO:2, a VH CDR3 of SEQ ID NO:3, a VL CDR1 of SEQ ID NO:4, a VL CDR2 of SEQ ID NO:5, a VL CDR3 of SEQ ID NO:6; (b) a VH CDR1 of SEQ ID NO:7, a VH CDR2 of SEQ ID NO:2, a VH CDR3 of SEQ ID NO:8, a VL CDR1 of SEQ ID NO:4, a VL CDR2 of SEQ ID NO:5, a VL CDR3 of SEQ ID NO:6; or (c) a VH CDR1 of SEQ ID NO:9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of GLL, a VL CDR1 of SEQ ID NO: 1 1 , a VL CDR2
  • Another embodiment provides an isolated antibody or antigen-binding portion thereof that specifically binds human SAS1 B, wherein said antibody binds the same human SAS1 B epitope recognized by a monoclonal antibody disclosed herein .
  • One embodiment provides an isolated antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion specifically binds human SAS1 B, wherein the antibody or antigen-binding portion thereof competes for binding with the antibody or antigen-binding portion thereof discussed herein.
  • Another embodiment provides an isolated antibody or antigen-binding portion thereof, wherein said antibody or antigen-binding portion thereof inhibits the binding of the isolated antibody or antigen-binding portion thereof of discussed to human SAS1 B.
  • the antibody or antigen-binding portion thereof specifically binds to a polypeptide consisting of amino acids 55-289 or 280-430 of SEQ ID NO: 1 12.
  • the antibody or antigen-binding portion thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a synthetic antibody, a single chain antibody, a diabody, or a CDR-grafted antibody.
  • the antibody or antigen-binding portion thereof comprises a VL amino acid sequence of SEQ ID NOs:76, 78, 80, 82, 84, 101 ,103, 105, 107, 109, or 1 1 1 .
  • the antibody or antigen-binding portion thereof comprises the VH amino acid sequence of SEQ ID NOs:75, 77, 79, 81 , 83, 100, 102, 104, 106, 108, or 1 10.
  • a composition comprising: (a) the antibody or antigen- binding portion thereof disclosed herein and a pharmaceutically acceptable carrier; or (b) the antibody or antigen-binding disclosed herein, wherein the antibody or antigen-binding portion thereof is conjugated to a therapeutic agent, and a pharmaceutically acceptable carrier.
  • ADC antibody-drug conjugate
  • the antibody or antigen-binding portion thereof specifically binds human SAS1 B with an affinity (Kd) of at least about 10 "6 M. In another embodiment, the said antibody or antigen-binding portion thereof binds to cancer cells.
  • An embodiment provides a radioimmunoconjugate comprising an antibody described herein linked to a radionuclide.
  • Another embodiment provides an isolated polypeptide consisting of one of SEQ ID NO: 1 -51 , 53-59, 75-84, or 100-1 1 1 or the isolated polynucleotide encoding said polypeptide.
  • One embodiment provides an isolated polynucleotide encoding an anti-human SAS1 B antibody or antigen-binding portion thereof, wherein said isolated polynucleotide encodes a heavy chain and a light chain, wherein: (a) the immunoglobulin heavy chain complementarity determining region (CDR) CDR1 comprises SEQ ID NO: 1 , 7, 9, 14, 25, 31 , 37, 43, 48, or 54, CDR2 comprises SEQ ID NO:2, 10, 15, 26, 32, 38, 44, 49, or 55, and CDR3 comprises SEQ ID NO:3, 8, 16, 27, 33, 39, 45, 50, 56, or GGL, and wherein the immunoglobulin light chain CDR1 comprises SEQ ID NO:4, 1 1 , 17, 28, 34, 40, 46, 51 , or 57, CDR2 comprises SEQ ID NO:5, 12, 18, 29, 35, 41 , 47, 52, or 58, and CDR3 comprises SEQ ID NO:6, 13, 19, 30, 36, 42, 53, or 59; or (b) the immunoglob
  • One embodiment provides a method for producing a human SAS1 B antibody or antigen-binding portion thereof, comprising culturing the isolated host cell of claim 19 and recovering said antibody.
  • the antibody is a chimeric antibody comprising VL and VH domains obtained from a mouse antibody, wherein said VL and VH domains comprise sequences capable of binding to human SAS1 B, and the VL and VH domains are fused to human CL and CH domains, respectively.
  • One embodiment provides a method of treating a hyperproliferative disorder comprising administering a composition disclosed herein to a mammal in need thereof.
  • Another embodiment provides a method of detecting a SASB1 polypeptide in a sample comprising: (a) contacting one or more antibodies disclosed with a test sample under conditions that allow polypeptide/antibody complexes to form; and (b) detecting polypeptide/antibody complexes; wherein the detection of polypeptide/antibody complexes is an indication that the human SAS1 B polypeptide is present in the sample.
  • One embodiment provides a method of detecting SAS I B-positive cells in a test sample comprising: (a) contacting one or more antibodies disclosed herein with the test sample under conditions that allow SAS1 B-positive cell/antibody complexes to form; and (b) detecting SAS1 B positive cell/antibody complexes; wherein the detection of SAS1 B positive cell/antibody complexes is an indication that SAS1 B cells are present in the test sample.
  • the sample is lymph node or tissue aspirate, serum, whole blood, cellular suspension, lymphocytes, whole blood, plasma, ovarian cyst fluid, pap smear, circulating tumor cells, tumor cells or tissue, ascites fluid, urine, or fluid effusion.
  • An embodiment provides antibodies and antigen-binding portions thereof that specifically bind to the human metallo-endoprotease SAS1 B, a product of the ASTL gene. These antibodies and antigen-binding portions thereof have applications in diagnostic assays to measure SAS1 B. They also have applications as therapeutic probes, both alone, as "naked” unconjugated antibodies, and conjugated with cytotoxic drugs and radionuclides. These antibodies also have therapeutic use as imaging agents.
  • Antibodies and antigen-binding fragments have been identified that are specific for at least two regions of human SAS1 B. These antibodies selectively recognize SAS1 B proteins in the presence of an array of other proteins. These antibodies can be used to precipitate SAS1 B from extracts of cells that express the SAS1 B protein, and can bind with SAS1 B in fixed and permeabilized cells.
  • Fig. 1 shows a diagram of the human SAS1 B polypeptide.
  • SAS1 B amino acid sequence is SEQ ID NO: 1 12.
  • Fig. 2 shows cell surface density of SAS1 B using RCT-7 antibody (also referred to herein as 2H2/2K5).
  • Fig. 3 shows a cell screening assay with RCT-7 antibody.
  • a polypeptide is a polymer of three or more amino acids covalently linked by amide bonds.
  • a polypeptide can be post-translationally modified.
  • a purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used in synthesis of the polypeptide, or combinations thereof.
  • a polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used in synthesis of the polypeptide has less than about 30%, 20%, 10%, 5%, 1 % or more of other polypeptides, culture medium, chemical precursors, and/or other chemicals used in synthesis. Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% or more pure.
  • a light or heavy chain variable region of an antibody has four framework regions interrupted by three hypervariable regions, known as complementary determining regions (CDRs). CDRs determine the specificity of antigen binding.
  • the heavy chain and light chain each have three CDRs, designated from the N terminus as CDR1 , CDR2, and CDR3 with the four framework regions flanking these CDRs.
  • the amino acid sequences of the framework region are highly conserved and CDRs can be transplanted into other antibodies. Therefore, a recombinant antibody can be produced by combining CDRs from one or more antibodies with the framework of one or more other antibodies.
  • antibodies include antibodies that comprise at least one, two, three, four, five, or six (or combinations thereof) of the CDRs of any of the monoclonal antibodies isolated from the hybridomas shown in Table 1 , or variant CDRs.
  • Variant CDRs are CDRs comprising amino acid sequences similar to the amino acid sequences of CDRs of any of the monoclonal antibodies produced by the hybridomas shown in Table 1 .
  • variant CDRs specifically bind to amino acids 55-289 or 280-430 of SEQ ID NO: 1 12 when present in an appropriate antibody structure (e.g., framework regions and other appropriate CDRs).
  • Polypeptides can comprise full-length human, mouse, or rabbit anti-SAS1 B heavy chain variable regions, full-length human, mouse or rabbit light chain regions, fragments thereof, and combinations thereof.
  • SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO: 4 SEQ ID NO:6
  • SEQ ID NO:7 SEQ ID NO:2 SEQ ID NO:8 SEQ ID NO: 4 SEQ ID NO:6
  • SEQ ID NO:31 SWAKG SEQ ID NO:33 SEQ ID NO:3 4 SEQ ID SEQ ID NO:36
  • SEQ ID NO:37 ASWKG SEQ ID NO:39 SEQ ID NO: 4 0 SEQ ID SEQ ID NO: 4 2
  • SEQ ID NO: 4 3 WAKG SEQ ID NO: 4 5 SEQ ID NO: 4 6 SEQ ID SEQ ID NO: 4 2
  • SEQ ID NO: 4 8 SWAKG SEQ ID NO:50 SEQ ID NO:51 SEQ ID SEQ ID NO:53 SEQ ID NO:35
  • An antibody can comprise a VH (variable heavy chain) of SEQ ID NOs:75, 77, 79, 81 , 83, 100, 102, 104, 106, 108, or 1 10.
  • An antibody can comprise a VL (variable light chain) of SEQ ID NOs:76, 78, 80, 82, 84, 101 , 103, 105, 107, 109, or 11 1.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:1 , 7, 9, 14, 20, 25, 31 , 37, 43, 48, or 54.
  • An antibody can comprise a VH CDR 2 of SEQ ID NOs:2, 10, 15, 21 , 26, 32, 38, 44, 49, or 55.
  • An antibody can comprise a VH CDR 3 of SEQ ID NOs:3, 8, 16, 22, 27, 33, 39, 45, 50, 56 or GGL.
  • An antibody can comprise a VL CDR 1 of SEQ ID NOs:4, 1 1 , 17, 23, 28, 34, 40, 46, 51 , or 57.
  • An antibody can comprise a VL CDR 2 of SEQ ID NOs:5, 12, 18, 29, 35, 41 , 47, 35, 58, or FAS.
  • An antibody can comprise a VL CDR3 of SEQ ID NOs:6, 13, 19, 24, 30, 36, 42, 42, 53, or 59.
  • An antibody can comprise a VH nucleic acid sequence of SEQ ID NO:60, 62, 64, 67, 69, 88, 90, 92, 94, 96, or 98.
  • An antibody can comprise a VL nucleic acid sequence of SEQ ID NO:61 , 63, 65, 68, 70, 89, 91 , 93, 95, 97, or 99.
  • An antibody can comprise a VH of SEQ ID NO:75 and a VL of SEQ ID NO:76.
  • An antibody can comprise a VH of SEQ ID NO:77 and a VL of SEQ I D NO:78.
  • An antibody can comprise a VH of SEQ ID NO:79 and a VL of SEQ I D NO:80.
  • An antibody can comprise a VH of SEQ ID NO:81 and a VL of SEQ I D NO:82.
  • An antibody can comprise a VH of SEQ ID NO:83 and a VL of SEQ ID NO:84.
  • An antibody can comprise a VH of SEQ ID NO: 100 and a VL of SEQ ID NO: 101 .
  • An antibody can comprise a VH of SEQ ID NO: 102 and a VL of SEQ ID NO: 103.
  • An antibody can comprise a VH of SEQ ID NO: 104 and a VL of SEQ ID NO: 105.
  • An antibody can comprise a VH of SEQ ID NO: 106 and a VL of SEQ ID NO: 107.
  • An antibody can comprise a VH of SEQ ID NO: 108 and a VL of SEQ ID NO: 109.
  • An antibody can comprise a VH of SEQ ID NO: 1 10 and a VL of SEQ ID NO: 1 1 1 .
  • An antibody can comprise a VH CDR1 of SEQ ID NO: 1 , a VH CDR2 of SEQ ID NO:2, and a VH CDR3 of SEQ ID NO:3.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:4, a VL CDR2 of SEQ ID NO:5, and a VL CDR3 of SEQ ID NO:6.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:7, a VH CDR2 of SEQ ID NO:2, and a VH CDR3 of SEQ ID NO:8.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:9, a VH CDR2 of SEQ ID NO: 10, and a VH CDR3 of GGL.
  • An antibody can comprise a VL CDR1 of SEQ I D NO: 1 1 , a VL CDR2 of SEQ ID NO: 12, and a VL CDR3 of SEQ ID NO: 13.
  • An antibody can comprise a VH CDR1 of SEQ ID NO: 14, a VH CDR2 of SEQ ID NO: 15, and a VH CDR3 of SEQ ID NO:16.
  • An antibody can comprise a VL CDR1 of SEQ I D NO: 17, a VL CDR2 of SEQ ID NO: 18, and a VL CDR3 of SEQ ID NO: 19.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:20, a VH CDR2 of SEQ ID NO:21 , and a VH CDR3 of SEQ ID NO:22.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:23, a VL CDR2 of FAS, and a VL CDR3 of SEQ ID NO:24.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:25, a VH CDR2 of SEQ ID NO:26, and a VH CDR3 of SEQ ID NO:27.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:28, a VL CDR2 of SEQ ID NO:29, and a VL CDR3 of SEQ ID NO:30.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:31 , a VH CDR2 of SEQ ID NO:32, and a VH CDR3 of SEQ I D NO:33.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:34, a VL CDR2 of SEQ ID NO:35, and a VL CDR3 of SEQ ID NO:36.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:37, a VH CDR2 of SEQ ID NO:38, and a VH CDR3 of SEQ ID NO:39.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:40, a VL CDR2 of SEQ ID NO:41 , and a VL CDR3 of SEQ ID NO:42.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:43, a VH CDR2 of SEQ ID NO:44, and a VH CDR3 of SEQ ID NO:45.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:46, a VL CDR2 of SEQ ID NO:47, and a VL CDR3 of SEQ ID NO:42.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:48, a VH CDR2 of SEQ ID NO:49, and a VH CDR3 of SEQ ID NO:50.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:51 , a VL CDR2 of SEQ ID NO:35, and a VL CDR3 of SEQ ID NO:53.
  • An antibody can comprise a VH CDR1 of SEQ ID NO:54, a VH CDR2 of SEQ ID NO:55, and a VH CDR3 of SEQ ID NO:56.
  • An antibody can comprise a VL CDR1 of SEQ ID NO:57, a VL CDR2 of SEQ ID NO:58, and a VL CDR3 of SEQ ID NO:59.
  • An antibody can have any of the above VHs combined with any of the above VLs.
  • An antibody can have any combination of VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, VL CDR3, variant VH CDR1 , variant VH CDR2, variant VH CDR3, variant VL CDR1 , variant VL CDR2, or variant VL CDR3.
  • an antibody comprises a VH of SEQ ID NOs:75, 77, 79, 81 , 83, 100, 102, 104, 106, 108, or 1 10 (or a variant thereof) and at least one, two or three VL CDRs of SEQ ID NOs:4, 5, 6, 1 1 , 12, 13, 17, 18, 19, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41 , 42, 46, 47, 51 , 35, 53, 57, 58, 59, FAS (or a variant thereof).
  • an antibody comprises a VL of SEQ ID NOs:76, 78, 80, 82, 84, 101 , 103, 105, 107, 109, 1 1 1 (or a variant thereof) and at least one, two or three VH CDRs of SEQ ID NOs: 1 , 2, 3, 7, 8, 9, 10, 14, 15, 16, 20, 21 , 22, 25, 26, 27, 31 , 32, 33, 37, 38, 39, 43, 44, 45, 48, 49, 50, 54, 55, 56, GGL (or a variant thereof).
  • An antibody can comprise the variable heavy chain CDRs from antibody 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1 /31 K1 .
  • An antibody can comprise the variable light chain CDRs from antibody 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1 /7K3, 9H1 /9K5, or 31 H1 /31 K1 .
  • An antibody can comprise a variable light chain that comprises the amino acid sequence of at least one or at least two or at least 3 CDRs of the 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1 /9K5, or 31 H1 /31 K1 antibody variable light chains.
  • An antibody can comprise a variable heavy chain that comprises the amino acid sequence of at least one or at least two or at least 3 CDRs of the 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H 1/31 K1 antibody variable heavy chains.
  • Heavy chain CDRs can be combined with appropriate variable regions of an antibody light chain.
  • Light chain CDRs optionally combined with heavy chain CDRs are, for example, CDRs comprising SEQ ID NOs: 4, 5, 6, 1 1 , 12, 13, 17, 18, 19, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41 , 42, 46, 47, 51 , 35, 53, 57, 58, 59, FAS, or CDRs functionally equivalent to these CDRs.
  • the respective amino acid sequences correspond to CDR1 (SEQ ID NOs:4, 1 1 , 17, 23, 28, 34, 40, 46, 51 , 57), CDR2 (SEQ ID NOs:5, 12, 18, 29, 35, 41 , 47, 52, 58, FAS), and CDR3 (SEQ ID NO:6, 13, 19, 24, 30, 36, 42, 53, 59) of an antibody light chain.
  • these light chain CDRs may be used independently of the heavy chains described above.
  • the CDRs are substituted for the corresponding CDR1 , CDR2, and CDR3, between the framework of a desired light chain variable region.
  • Light chain CDRs can be combined with appropriate variable regions of an antibody heavy chain.
  • Heavy chain CDRs optionally combined with light chain CDRs are, for example, CDRs comprising SEQ ID NOs: 1 , 2, 3, 7, 8, 9, 10, 14, 15, 16, 20, 21 , 22, 25, 26, 27, 31 , 32, 33, 37, 38, 39, 43, 44, 45, 48, 49, 50, 54, 55, 56, GGL, or CDRs functionally equivalent to these CDRs.
  • the respective amino acid sequences correspond to CDR1 (SEQ ID NO: 1 , 7, 9, 14, 20, 25, 31 , 37, 43, 48, 54), CDR2 (SEQ ID NO:2, 10, 15, 21 , 26, 32, 38, 44, 49, 55), and CDR3 (SEQ ID NOs:3, 8, 16, 22, 27, 33, 39, 45, 50, 56, GGL) of an antibody light chain.
  • these heavy chain CDRs may be used independently of the light chains described above.
  • the CDRs are substituted for the corresponding CDR1 , CDR2, and CDR3 regions, between the framework of a desired heavy chain variable region.
  • a polypeptide variant or variant CDR differs by about, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 or more amino acid residues (e.g., amino acid additions, substitutions or deletions) from a polypeptide shown in SEQ ID NOs: 1 -51 , 53-59, 75-84, 100-1 1 1 or a fragment thereof. Where this comparison requires alignment the sequences are aligned for maximum homology. The site of variation can occur anywhere in the polypeptide.
  • a variant polypeptide has activity substantially similar to a polypeptide shown in SEQ ID NOs: 1 -51 , 53-59, 75-84, and 100-1 1 1 .
  • Activity substantially similar means that when the polypeptide is used to construct an antibody, the antibody has the same or substantially the same activity as an antibody shown in Table 1 .
  • the variant polypeptides can have conservative amino acid substitutions at one or more predicted non-essential amino acid residues.
  • a conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • the following groups of amino acids represent conservative changes: (1 ) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • a variant polypeptide can also be isolated using a hybridization technique. Briefly, DNA having a high homology to the whole or part of a nucleic acid molecule of SEQ ID NOs:60-65, 67-70, 88-99 or a nucleic acid molecule encoding a polypeptide shown in SEQ ID NOs: 1 -51 , 53-59, 75-84, or 100-1 1 1 is used to prepare a polypeptide.
  • a polypeptide also includes polypeptides that are variants of SEQ ID NOs: 1 -51 , 53-59, 75-84, 100-1 1 1 , and polypeptides that are encoded by a nucleic acid molecule that hybridizes under high stringency with a nucleic acid molecule encoding SEQ ID NOs:60-65, 67-70, 88-99, or a complement thereof.
  • nucleic acid sequences that encode polypeptides using readily available codon tables. As such, these nucleic acid sequences are not presented herein.
  • Identity or identical means amino acid sequence (or nucleic acid sequence) similarity and has an art recognized meaning. Sequences with identity share identical or similar amino acids (or nucleic acids). Sequence identity is the percentage of amino acids identical to those in the antibody's original amino acid sequence, determined after the sequences are aligned and gaps are appropriately introduced to maximize the sequence identity as necessary. Thus, a candidate sequence sharing 85% amino acid sequence identity with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 85% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence, and/or constitute conservative amino acid changes.
  • Antibodies can comprise CDRs of 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1/31 K1 antibodies or variant antibodies comprising one or more variant CDRs.
  • These variant antibodies can have an activity equivalent (e.g., binding to human SAS1 B with the same or substantially similar Kd as an antibody produced by a hybridoma of Table 1 ) to that of 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1 /31 K1 .
  • an activity equivalent e.g., binding to human SAS1 B with the same or substantially similar Kd as an antibody produced by a hybridoma of Table 1 .
  • Antibody variants retain substantially the same functional activity of 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1/31 K1 antibodies.
  • Naturally-occurring functionally active variant antibodies such as allelic variants and species variants and non-naturally occurring functionally active variants are included herein and can be produced by, for example, mutagenesis techniques or by direct synthesis.
  • Antibody variants are encoded by variant polypeptides and variant CDRs of SEQ I D NOs: 1 -51 , 53-59, 75-84, and 100-111.
  • Polypeptide variants orCDR variants of SEQ ID NOs: 1-51, 53-59, 75-84, 100- 111 are part of this disclosure.
  • Polypeptide variants or CDR variants of SEQ ID NOs: 1-51, 53-59, 75-84, 100-111 can comprise one or more amino acid substitutions, additions or deletions.
  • a variant polypeptide or variant CDR includes an amino acid sequence at least about 75% identical to a sequence shown as SEQ ID NOs:1-51, 53-59, 75-84, and 100-111.
  • the variant polypeptide or CDR is at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more identical to SEQ ID NOs: 1-51, 53-59, 75-84, and 100-111.
  • Variant polypeptides or variant CDRs encode a variant antibody, which is an antibody comprising an amino acid sequence of SEQ ID NOs:1-51, 53-59, 75-84, 100-111 in which one or more amino acid residues have been added, substituted or deleted.
  • the variable region of an antibody can be modified to improve its biological properties, such as antigen binding. Such modifications can be achieved by e.g., site-directed mutagenesis, PCR-based mutagenesis, cassette mutagenesis.
  • Variant antibodies comprise an amino acid sequence which is at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more identical to the amino acid sequence of a heavy or light chain variable region of 7H2, 6C1, 1H2/1K2, 1H3/1K3, 2H2/2K5, 6B1, 3F2, 3H4/3K5, 5H2/5K1, 6H2/6K1, 7H1/7K3, 9H1/9K5, or 31H1/31K1.
  • a variant antibody retains the same function of a 7H2, 6C1, 1H2/1K2, 1H3/1K3, 2H2/2K5, 6B1, 3F2, 3H4/3K5, 5H2/5K1, 6H2/6K1, 7H1/7K3, 9H1/9K5, or 31 H1/31 K1 antibody (e.g., binds human SAS1B, in particular a C-terminal region of human SAS1B such as amino acids 280-430 of SEQ ID NO:112) or at an N-terminal region of human SAS1 B such as amino acids 55-289 of SEQ ID NO:112 at the same or substantially similar Kd as an antibody produced by the hybridomas shown in Table 1, e.g.
  • a variant antibody may have a function that is somewhat altered from a 7H2, 6C1, 1H2/1K2, 1H3/1K3, 2H2/2K5, 6B1, 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or31H1/31K1 antibody (e.g., binding human SAS1 B with a K d that is higher or lower than an 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1/31 K1 antibody).
  • Polypeptide sequences can be modified, for example, by synthesizing multiple polynucleotides encoding the amino acid sequence of a variable region, and preparing nucleic acids encoding the variable region by PCR using the polynucleotides.
  • Antibodies that comprise one or more CDRs can be prepared by inserting the polynucleotide into an appropriate expression vector and expressing the polynucleotide.
  • polynucleotides can be synthesized using mixed nucleotides to prepare a DNA library that encodes a variety of antibodies comprising CDRs with various amino acids introduced at certain positions.
  • An antibody can be isolated by selecting from the library a clone encoding an antibody that binds to human SAS1 B with a Kd that is the same or substantially similar to the Kd of an antibody produced by a hybridoma shown in Table 1 .
  • a polypeptide or antibody can be covalently or non-covalently linked to an amino acid sequence to which the polypeptide or antibody is not normally associated with in nature. Additionally, a polypeptide or antibody can be covalently or non- covalently linked to compounds or molecules other than amino acids.
  • a polypeptide or antibody can be linked to an indicator reagent, an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, or a combination thereof.
  • a protein purification ligand can be one or more C amino acid residues at, for example, the amino terminus or carboxy terminus of a polypeptide.
  • An amino acid spacer is a sequence of amino acids that are not usually associated with a polypeptide or antibody in nature. An amino acid spacer can comprise about 1 , 5, 10, 20, 100, or 1 ,000 amino acids.
  • a polypeptide can be a fusion protein, which can also contain other amino acid sequences, such as amino acid linkers, amino acid spacers, signal sequences, TMR stop transfer sequences, transmembrane domains, as well as ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A, or combinations thereof.
  • a fusion protein is two or more different amino acid sequences operably linked to each other.
  • a fusion protein construct can be synthesized chemically using organic compound synthesis techniques by joining individual polypeptide fragments together in fixed sequence.
  • a fusion protein construct can also be expressed by a genetically modified host cell (such as E.
  • heterologous polypeptide can be fused, for example, to the N-terminus or C-terminus of a polypeptide.
  • a polypeptide can also comprise homologous amino acid sequences, i.e., other immunoglobulin-derived sequences. More than one polypeptide can be present in a fusion protein. Fragments of polypeptides can be present in a fusion protein.
  • a fusion protein can comprise, e.g., one or more of SEQ ID NOs: 1 -51 , 53-59, 75-84, 100-1 1 1 , fragments thereof, or combinations thereof.
  • Polypeptides can be in a multimeric form. That is, a polypeptide can comprise two or more copies of SEQ ID NOs: 1 -51 , 53-59, 75-84, 100-1 1 1 or a combination thereof.
  • a polypeptide is derived from a human, rabbit, mouse, other mammal, or combinations thereof.
  • a polypeptide can be isolated from cells or tissue sources using standard protein purification techniques.
  • Polypeptides can also be synthesized chemically or produced by recombinant DNA techniques.
  • a polypeptide can be synthesized using conventional peptide synthesizers.
  • a polypeptide can be produced recombinantly.
  • a polynucleotide encoding a polypeptide can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art.
  • a suitable expression host cell system A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used.
  • a polynucleotide encoding a polypeptide can be translated in a cell-free translation system.
  • antibodies refers to an intact antibody or an antigen-binding portion or fragment thereof that competes with the intact antibody for antigen binding.
  • antibodies also includes any type of antibody molecule or specific binding molecule that specifically binds SAS1 B.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide, glycoprotein or immunoglobulin that specifically binds SAS1 B to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of nucleic acids encoding antibody variable and optionally constant domains.
  • An antibody can be any isotype including IgG (lgG1 , lgG2, lgG2a, Ig2b, lgG3, lgG4), IgM, IgA (lgA1 and lgA2), IgD, and IgE.
  • a monoclonal antibody is an antibody obtained from a group of substantially homogeneous antibodies.
  • a group of substantially homogeneous antibodies can contain a small amount of mutants or variants.
  • Monoclonal antibodies are highly specific and interact with a single antigenic site. Each monoclonal antibody typically targets a single epitope, while polyclonal antibody populations typically contain various antibodies that target a group of diverse epitopes.
  • Monoclonal antibodies can be produced by many methods including, for example, hybridoma methods (Kohler and Milstein, Nature 256:495, 1975), recombination methods (U.S. Pat. No.
  • a “humanized antibody or antigen-binding fragment” thereof is an antibody or fragment thereof that has been engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rabbit, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and/or light chain.
  • CDRs complementarity-determining regions
  • a humanized antibody comprises sequences that are entirely human except for the CDR regions.
  • Humanized antibodies are typically less immunogenic to humans, relative to non-humanized antibodies, and thus offer therapeutic benefits in certain situations.
  • a "human antibody or antigen binding fragment thereof” is an antibody or antigen binding fragment thereof that contains only human-derived amino acid sequences.
  • a fully human antibody may be produced from a human B- cell or a human hybridoma cell.
  • the antibody may be produced from a transgenic animal that contains the locus for a human heavy chain immunoglobulin and a human light chain immunoglobulin, or contains a nucleic acid that encodes the heavy and light chains of a specific human antibody.
  • a human antibody or antigen binding fragment thereof is still considered a "human antibody or antigen binding fragment thereof" even if the framework and/or CDRs of the heavy chain variable domain or light chain variable domain of the antibody isolated or obtained from a human cell, human cell line, or other methodology are mutated (e.g., by amino acid substitution(s), addition(s), and/or deletion(s)) to improve the affinity or other properties of the antibody.
  • the human antibody isolated or obtained from a human cell or human cell line is mutated so that it has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the amino acid sequence of the antibody isolated or obtained from a human cell or human cell line.
  • six, five, four, three, two, or one amino acid substitutions are made in one, two, three, four, five, and/ or six of the CDRs.
  • a human antibody has an amino acid sequence that is substantially identical to an antibody isolated or obtained from a human cell or human cell line, but is not naturally occurring.
  • the non-naturally occurring human antibody has one or more mutations in the amino acid sequence that do not occur in the variable heavy or light CDR regions, and do not affect the binding or therapeutic characteristics of the human antibody.
  • Chimeric antibodies or antigen-binding portions thereof have a part of a heavy chain and/or light chain that is derived from a specific species or a specific antibody class or subclass, and the remaining portion of the chain is derived from another species, or another antibody class or subclass. See e.g., Morrison, Science 229: 1202 (1985); Oi et ai, BioTechniques 4:214 (1986); Gillies et ai, J. Immunol. Methods 125: 191 -202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.
  • Chimeric antibodies can be produced using a variety of techniques including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530, 101 ; and 5,585,089), veneering or resurfacing (EP 592, 106; EP 519,596; Padlan, Molecular Immunology 28:489-498 (1991 ); Studnicka et ai, Protein Engineering 7(6):805-814 (1994); Roguska et ai, PNAS 96:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
  • CDR-grafting EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530, 101 ; and 5,585,089)
  • veneering or resurfacing EP 592, 106; EP 519,596; Padlan, Molecular Immunology 28:4
  • a chimeric antibody can comprise variable and constant regions of species that are different from each other, for example, an antibody can comprise the heavy chain and light chain variable regions of a non-human mammal such as a mouse or rabbit, and the heavy chain and light chain constant regions of a human.
  • an antibody can be obtained by ligating a polynucleotide encoding a variable region of a mouse or rabbit antibody to a polynucleotide encoding a constant region of a human antibody; incorporating the ligated polynucleotides into an expression vector; and introducing the vector into a host cell for production of the antibody. See WO 96/02576.
  • the host cells can be eukaryotic cells, such as mammalian cells, including, e.g., CHO cells, lymphocytes, and myeloma cells.
  • the chimeric antibody can comprise additional amino acid acids that are not included in the CDRs introduced into the recipient antibody, nor in the framework sequences. These amino acids can be introduced to more accurately optimize the antibody's ability to recognize and bind to an antigen. For example, as necessary, amino acids in the framework region of an antibody variable region can be substituted such that the CDR of a reshaped antibody forms an appropriate antigen-binding site. See Sato et ai, Cancer Res. (1993) 53:851 -856.
  • Non-limiting examples of antigen-binding fragments of antibodies include: Fab fragments; Fab' fragments, Fab'-SH fragments, F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; sdAb fragments (nanobodies); Fab- like antibodies (an antigen-binding fragment containing variable regions of a heavy chain and light chain that is equivalent to Fab fragments that are obtained by papain digestion); F(ab')2-like antibodies (an antigen-binding fragment containing two antigen-binding domains that is equivalent to F(ab')2 fragments that are obtained by pepsin digestion), multispecific antibodies prepared from antibody fragments, diabody, bispecific antibody, multifunctional antibody, chimeric antibody, humanized antibody, human antibody, murine antibody, rabbit antibody synthetic antibody, CDR- grafted antibody, and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementar
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g, monovalent nanobodies, bivalent nanobodies), single-chain (Fv)2 (sc(Fv)2); divalent (sc(Fv)2); tetravalent ([sc(Fv)2]2) scFV antibodies, and small modular immunopharmaceuticals (SMI Ps), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
  • SMI Ps small modular immunopharmaceuticals
  • variable domain An antigen-binding fragment of an antibody will typically comprise at least 1 variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least 1 , 2 or 3 CDRs, which are adjacent to or in frame with 1 , 2, 3, or 4 framework sequences, in antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • a “diabody” is a bivalent minibody constructed by gene fusion (see, e.g., Holliger et ai, Proc. Natl. Acad. Sci. U. S. A., 90:6444 (1993); EP 404,097; WO 93/1 1 161 ).
  • Diabodies are dimers composed of two polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers. The number of amino acid residues that constitute a linker can be between about 2 to 12 residues (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12).
  • the linkers of the polypeptides in a diabody are typically too short to allow the VL and VH to bind to each other.
  • Diabody technology provides an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • a scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see e.g., Huston et ai, PNAS USA, 85:5879 (1988); Pluckthun, "The Pharmacology of Monoclonal Antibodies” Vol.1 13, Ed Resenburg & Moore, Springer Verlag, New York, pp.269-315, (1994)).
  • the order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: VH-linker-VL; or VL-linker-VH.
  • the H chain V region and L chain V region in a scFv may be derived from any anti-SAS1 B antibody or antigen- binding fragment thereof described herein.
  • a sc(Fv)2 is a fragment where two VHs and two VLs are linked by a linker to form a single chain (Hudson et ai, J. Immunol. Methods, 231 : 177 (1999)).
  • a sc(Fv)2 molecule can be prepared, for example, by connecting scFvs with a linker.
  • sc(Fv)2 molecules can include antibodies where two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL (VH- linker-VL-linker-VH-linker-VL), beginning from the N terminus of a single-chain polypeptide; however the order of the two VHs and two VLs is not limited to this arrangement, and they may be arranged in any order. Examples of arrangements are listed below:
  • linkers are usually required when four antibody variable regions are linked; the linkers used may be identical or different. There is no limitation on the linkers that link the VH and VL regions of the antibody fragments.
  • the linker is a peptide linker. Any arbitrary single-chain peptide comprising about three to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18 amino acids) can be used as a linker.
  • peptide linkers include: Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 1 13); Ser Gly Gly Gly (SEQ ID NO: 1 14); Gly Gly Gly Gly Ser (SEQ ID NO: 1 15); Ser Gly Gly Gly Gly (SEQ ID NO: 1 16); Gly Gly Gly Gly Ser (SEQ I D NO: 1 17); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 1 18); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 1 19); Ser Gly Gly Gly Gly Gly (SEQ ID NO: 120); (Gly Gly Gly Ser (SEQ ID NO: 121 ) n , wherein n is an integer of one or more; and (Ser Gly Gly Gly (SEQ ID NO:52) n , wherein n is an integer of one or more.
  • the linker is a synthetic compound linker (chemical cross- linking agent).
  • cross-linking agents include, for example, N- hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2 (sulfo)
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the SAS1 B protein. Other such antibodies may combine an SAS1 B binding site with a binding site for another protein. Bispecific antibodies can be prepared as full length antibodies or low molecular weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies). Full length bispecific antibodies can be produced based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)).
  • bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods.
  • Antibodies can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • An exemplary dimerization domain comprises an Fc region or a hinge region.
  • a multivalent antibody can comprise about 3, 4, 5, 6, 7, 8, or more antigen binding sites.
  • the multivalent antibody optionally comprises at least one, two, three or more polypeptide chains, wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VDI-(XI) n - VD2-(X2) n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or peptide spacer, and n is 0 or 1 .
  • binding molecules e.g., antibodies or antigen- binding fragments
  • the antibodies or antigen-binding fragments thereof specifically bind to an epitope within the C-terminal domain (e.g., amino acids 280-430 of human SAS1 B (SEQ ID NO: 1 12) or with the N-terminal domain (e.g., amino acids 55-289 of SEQ ID NO: 1 12).
  • the antibodies or antigen-binding fragments thereof specifically bind to an epitope within amino acids of 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390- 400, 400-410, 410-420, 420-430, 280-310, 310-340, 340-370, 370-400, 400-430, 280-300, or 300 to 430 of SEQ ID NO: 1 12 or combinations thereof.
  • the antibodies or antigen-binding fragments thereof specifically bind to an epitope within amino acids 55-65, 65-75, 75-85, 85-95, 95-105, 105-1 15, 1 15- 125, 125-135, 135-145, 145-155, 155-165, 165-175, 175-185, 185-195, 195-205, 205-215, 225-235, 235-245, 245-255, 255-265, 265-275, 275-289, 55-85, 85-1 15, 1 15-145, 145-175, 175-205, 205-235, 235-265, 265-289, or 80-163 of SEQ ID NO: 1 12 or combinations thereof.
  • the antibody or antigen-binding portion thereof may bind to conformational epitope which comprises 2 or more of these regions.
  • An antibody or fragment thereof binds to an epitope that overlaps with or is the same (i.e., a substantially identical epitope) as any of the monoclonal antibodies shown in Table 1 .
  • An antibody that binds to an epitope substantially identical to an epitope of human SAS1 B to which a monoclonal antibody of Table 1 binds can be obtained by analyzing epitopes of the monoclonal antibodies of Table 1 using well known epitope mapping methods.
  • Competitive assays can be used to determine if two antibodies bind to a substantially identical epitope of SAS1 B.
  • the first antibody and the second antibody can be considered to bind to a substantially identical epitope on SAS1 B.
  • Competitively inhibits means that an antibody or antigen-binding fragment thereof can specifically bind an epitope that a monoclonal antibody produced by a hybridoma cell line shown in Table 1 is directed to, using conventional reciprocal antibody competition assays. See e.g., Belanger et al. (1973), Clinica Chimica Acta 48: 15.
  • Antibodies that competitively inhibit binding of one or more of 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, or 31 H1 /31 K1 or antigen-binding fragments thereof reduce the binding of one or more of 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1 /7K3, 9H1/9K5, or 31 H1/31 K1 or antigen-binding fragments thereof to a SAS1 B polypeptide (e.g., a full-length SAS1 B polypeptide or amino acids 280-430 or amino acids 55-289 of SEQ ID NO: 1 12) or to cancer cells by about 40%, 50%, 75%, 90%
  • Antibodies and antigen-binding fragments thereof can inhibit the binding 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1 /9K5, or 31 H1/31 K1 to human SAS1 B.
  • antibodies that bind to an epitope that is substantially identical to or the same as an epitope of SAS1 B to which an antibody produced by a hybridoma of Table 1 binds, and that can also comprise the activity of binding to SAS1 B or fragments thereof (e.g. amino acids 280-430 or 55-289 of SEQ ID NO: 1 12) are disclosed herein.
  • Antibodies can specifically bind SAS1 B (e.g. human SAS1 B).
  • “Specifically binds” means that the antibody recognizes and binds to SAS1 B with greater affinity than to other, non-specific molecules that are not SAS1 B.
  • an antibody raised against an antigen (polypeptide) to which it binds more efficiently than to a non-specific antigen e.g., a protein that is not related to or homologous to SAS1 B
  • a non-specific antigen e.g., a protein that is not related to or homologous to SAS1 B
  • Binding specificity can be tested using, for example, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay using methodology well known in the art.
  • Antibodies, antigen-binding fragments thereof, or variants thereof can specifically bind SAS1 B with a wide range of disassociation constants (Kd).
  • Kd disassociation constants
  • an antibody can bind human SAS1 B with a Kd equal to or less than about 10 "7 M, such as but not limited to, 0.1 -9.9 x 10 "5 , 10 “6 , 10 “7 , 10 “8 , 10 “9 , 10 “10 , 10 "11 , 10 " 12 1 Q- 13 , 1 Q- 14 , 1 Q- 15 or an y ran g e or value therein, as determined by e.g., surface plasmon resonance or the Kinexa method.
  • An embodiment encompasses antibodies that bind human SAS1 B polypeptides with a disassociation constant or Kd that is within any one of the ranges that are between each of the individual recited values.
  • An antibody has the same or substantially identical activity as antibodies produced by the hybridomas shown in Table 1 when the Kd for binding to SAS1 B (e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12) is within about 0.1 .
  • Antibodies, antigen-binding fragments thereof or variants thereof can specifically bind human SAS1 B polypeptides with an off rate (K 0 ff) of less than or equal to 01 .-9.9 x 10 "3 sec 1 , 10 "4 sec 1 , 10 "5 sec 1 , 10 “6 sec “1 , 10 “7 sec 1 .
  • An embodiment encompasses antibodies that specifically bind SAS1 B polypeptides with an off rate that is within any one of the ranges that are between each of the individual recited values.
  • An antibody has the same or substantially identical activity as antibodies produced by the hybridomas shown in Table 1 when the K 0 ff for binding to SAS1 B (e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12) is within about 0.1 . , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% (or any range or particular value between 0.1 and 20%) of the K 0 ff for binding to SAS1 B (e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12) of an antibody produced by the hybridomas shown in Table 1 .
  • the K 0 ff for binding to SAS1 B e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12
  • Antibodies, antigen-binding fragments thereof, or variants thereof can specifically bind SAS1 B polypeptides with an on rate (K on ) greater than or equal to 0.1 -9.9 x 10 3 M- 1 sec- ⁇ 10 4 IV sec “1 , 10 5 IV sec “1 , 10 6 IV sec “1 , 10 7 IV sec “1 , 10 8 M “ 1 sec _1 .
  • An embodiment encompasses antibodies that bind SAS1 B polypeptides with on rate that is within any one of the ranges that are between each of the individual recited values.
  • An antibody has the same or substantially identical activity as antibodies produced by the hybridomas shown in Table 1 when the K on for binding to SAS1 B (e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12) is within about 0.1 . , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20% (or any range or particular value between 0.1 and 20%) of the K on for binding to SAS1 B (e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12) of an antibody produced by the hybridomas shown in Table 1 .
  • SAS1 B e.g., amino acids 55-289 or 280-430 of SEQ ID NO: 1 12
  • Antibodies can be produced using methods known to those of skill in the art.
  • an SAS1 B antigen or a fragment thereof e.g., amino acids 55-289 or fragment thereof can be conjugated to a carrier protein and/or administered to the animals with an adjuvant.
  • An SAS1 B antigen can comprise one or more epitopes (i.e., antigenic determinants).
  • An epitope can be a linear epitope, sequential epitope or a conformational epitope. Epitopes within a polypeptide can be identified by several methods. See, e.g., U.S. Patent No. 4,554, 101 ; Jameson & Wolf, CABIOS 4: 181 -186 (1988).
  • SAS1 B can be isolated and screened.
  • a series of short peptides which together span the entire SAS1 B polypeptide sequence, can be prepared by proteolytic cleavage.
  • each fragment can be tested for the presence of epitopes recognized in an ELISA.
  • an SAS1 B antigen such as a 100-mer polypeptide fragment
  • a solid support such as the wells of a plastic multi-well plate.
  • a population of antibodies are labeled, added to the solid support and allowed to bind to the unlabeled antigen, under conditions where non-specific absorption is blocked, and any unbound antibody and other proteins are washed away.
  • Antibody binding is detected by, for example, a reaction that converts a colorless substrate into a colored reaction product. Progressively smaller and overlapping fragments can then be tested from an identified 100-mer to map the epitope of interest.
  • Methods for preparing monoclonal antibodies from hybridomas are well known to those of skill in the art and include, e.g., standard cell culture methods and ascites production methods.
  • Recombinant antibodies or fragments thereof produced by gene engineering can be made using the polynucleotide sequences disclosed herein.
  • Genes encoding antibodies or fragments thereof can be isolated from hybridomas described herein or other hybridomas. The genes can be inserted into an appropriate vector and introduced into a host cell. See, e.g., Borrebaeck & Larrick, Therapeutic Monoclonal Antibodies, Macmillan Publ. Ltd, 1990.
  • Antibodies can be produced using immunospot array assay on a chip (ISAAC) to obtain an antibody gene by screening single B cells, which secrete a specific monoclonal antibody, within several weeks (Jin et al., 2009 Nat. Med. 15, 1088- 1092).
  • Whole antibodies can also be made using PCR primers having VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site to amplify the VH or VL sequences in scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., a human, rabbit or mouse constant region
  • the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., a human VL constant region or rabbit or murine light constant regions.
  • the vectors for expressing the VH or VL domains can comprise, e.g., a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker.
  • VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • nucleic acid sequences for human, rabbit, and mouse IgG constant regions have been cloned and sequenced and can be used to construct antibodies.
  • Human antibodies can be made by sensitizing human lymphocytes with antigens of interest or cells expressing antigens of interest in vitro; and fusing the sensitized lymphocytes with human myeloma cells.
  • a human antibody can be made by using an antigen to immunize a transgenic animal that comprises a partial or entire repertoire of human antibody genes.
  • Human antibodies can also be made by panning with a human antibody library.
  • the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of a phage, using phage display method, and phages that bind to the antigen are selected.
  • scFv single chain antibody
  • the polynucleotides encoding the variable regions of human antibodies that bind to the antigen can be determined. If the polynucleotide sequences of scFvs that bind to the antigen are identified, appropriate expression vectors comprising these sequences can be constructed, and then introduced into appropriate hosts and expressed to obtain human antibodies.
  • Antibodies and fragments thereof can be purified by any method, including, e.g., protein A-Sepharose methods, hydroxyapatite chromatography, salting-out methods with sulfate, ion exchange chromatography, affinity chromatography, filtration, ultrafiltration, dialysis, preparative polyacrylamide gel electrophoresis, isoelectrofocusing or combinations thereof.
  • Antibodies can be dried or lyophilized ("freeze-dried) for more ready formulation into a desired vehicle/carrier where appropriate and for increased shelf- life.
  • Antibodies can be covalently attached to other molecules such that covalent attachment does not affect the ability of the antibody to bind to SAS1 B or cells expressing SAS1 B.
  • antibodies can be modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups (e.g., methyl group, group ethyl group, carbohydrate group), proteolytic cleavage, linkage to a cellular ligand or other protein.
  • Conjugated antibodies can be bound to various molecules including, for example, polymers, hyaluronic acid, fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, cytotoxic agents, radionuclides, and drugs.
  • An anti-SAS1 B antibody or antigen-binding fragment thereof can be modified with a moiety that improves its binding, stabilization, and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1 .5, 2, 5, 10, or 50 fold.
  • the anti-SAS1 B antibody or antigen-binding fragment thereof can be associated with (e.g., conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide, a polyethylene oxide, polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers.
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., conjugated to
  • a polymer e.g., a substantially non-anti
  • Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1 ,000 to about 15,000, and 2,000 to about 12,500) can be used.
  • the anti-SAS1 B antibody or antigen-binding fragment thereof can be conjugated to a water soluble polymer, a hydrophilic polyvinyl polymer, polyvinylalcohol or polyvinylpyrrolidone.
  • polymers examples include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) (see e.g., Chapman et al., Nature Biotechnology, 17: 780 (1999), or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.
  • the antibodies or antigen-binding fragments thereof can also be conjugated to small molecules and other chemical moieties.
  • Conjugated antibodies can be prepared by performing chemical modifications on the antibodies or fragments thereof. See e.g., US 5057313 and US 5156840.
  • the constant region of an antibody or antigen-binding fragment thereof can be a human Fc region, e.g., a wild-type Fc region, or an Fc region that includes one or more amino acid substitutions.
  • the constant region can have substitutions that modify the properties of the antibody (e.g., increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
  • Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g. , Fc receptor binding and complement activation. For example, antibodies may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • Antibodies can also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of lgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30: 105-08). See also, e.g., U.S. 2005/0037000.
  • the amino acid sequence of the heavy chain variable region (VH) or the light chain variable region (VL) in the antibody or antibody fragments can include modifications such as amino acid substitutions, deletions, additions, and/or insertions.
  • the modification may be in one or more of the CDRs of the anti-SAS1 B antibody or antigen-binding fragment thereof.
  • the modification involves one, two, or three amino acid substitutions in one or more CDRs and/or framework regions of the VH and/or VL domain of the anti-SAS1 B antibody. Such substitutions are made to improve the binding, functional activity and/or reduce immunogenicity of the anti-SAS1 B antibody.
  • the amino acid substitutions can be conservative amino acid substitutions.
  • one, two, or three amino acids of the CDRs of the anti-SAS1 B antibody or antigen-binding fragment thereof may be deleted or added as long as there is SAS1 B binding and/or functional activity when VH and VL are associated.
  • the amino acid sequences of the CDRs are of primary importance for epitope recognition and antibody binding. Changes may be made to the amino acids that comprise the CDRs without interfering with the ability of the antibody to recognize and bind its cognate epitope. For example, changes that do not affect epitope recognition, yet increase the binding affinity of the antibody for the epitope may be made. Thus, also included in the scope of the present disclosure are improved versions of the disclosed antibodies, which also specifically recognize and bind SAS1 B, optionally with increased affinity.
  • equivalents of a primary antibody have been generated by changing the sequences of the heavy and light chain genes in the CDR1 , CDR2, CDR3, or framework regions, using methods such as oligonucleotide-mediated site- directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E. coli. See Vaughan et ai, 1998, Nature Biotechnology, 16: 535; Adey et ai, 1996, Chapter 16, pp. 277-291 , in "Phage Display of Peptides and Proteins", Eds. Kay et ai, Academic Press).
  • the antibody sequences described herein can be used to develop anti-SAS1 B antibodies with improved functions, including improved affinity for SAS1 B.
  • An embodiment encompasses antibodies, fragments thereof, or variants thereof that have one or more of the same or substantially similar biological characteristics as the antibodies shown in Table 1 .
  • Biological characteristics are the in vitro or in vivo activities or properties of the antibodies shown in Table 1 , including, for example, the ability to bind to SAS1 B (e.g., amino acids 280-430 or amino acids 55-289 of SEQ ID NO: 1 12) with a substantially similar K d , K off , and/or K on rate, bind cancer cells, cause death of cancer cells, or combinations thereof.
  • Antibodies can be used to generate anti-idiotype antibodies that "mimic" human SAS1 B polypeptides using techniques well known to those skilled in the art. See, Greenspan & Bona, FASEB 17:437-444 (1993); Nissinoff, J. Immunol. 147:2429-2438 (1991 ).
  • One embodiment provides mixtures of antibodies, antigen-binding fragments thereof, or variants thereof that bind to SAS1 B, wherein the mixture has at least two, three, four, five or more different antibodies.
  • An embodiment also provides for panels of antibodies that have different affinities for SASB1 , different specificities for SAS1 B, or different dissociation rates.
  • An embodiment provides panels of at least about 2, 3, 4, 5, 6, 7, 10, 20, 50, 100, 250, 500, 750, or 1 ,000 antibodies.
  • the antibodies or antigen-binding fragments thereof are not naturally occurring due to one or more amino acid mutations in one or more constant regions or one or more framework regions or other mutations.
  • Polynucleotides contain less than an entire human, mouse or rabbit genome and can be single- or double-stranded nucleic acids.
  • a polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof.
  • the polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides.
  • the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure by dry weight. Purity can be measured by a method such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • polynucleotides encode the polypeptides described above.
  • polynucleotides encode polypeptides shown in, e.g., SEQ ID NOs:60-65, 67-74, 88-99 or portions or combinations thereof.
  • polynucleotides encode the polypeptides and antibodies disclosed herein, as well as fragments thereof.
  • a polynucleotide fragment can be about 9, 18, 21 , 27, 30, 33, 39, 48, 51 , 75, 100, 120, 130, 140, 150, 200 or more polynucleotides.
  • One of skill in the art can obtain the polynucleotide sequences using the polypeptide sequences and codon tables known to those of skill in the art.
  • Polynucleotides can contain naturally occurring polynucleotides or sequences that differ from those of any naturally occurring sequences or polynucleotides (e.g., non-naturally occurring polynucleotides).
  • Polynucleotides can differ from naturally occurring nucleic acids, but still encode naturally occurring amino acids due to the degeneracy of the genetic code. Polynucleotides can also comprise other heterologous nucleotide sequences, such as sequences coding for linkers, signal sequences, amino acid spacers, heterologous signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands useful in protein purification such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. Polynucleotides can also comprise other nucleotide sequences.
  • polynucleotides e.g., vectors comprising the polynucleotides or naked polynucleotides
  • transformation methods using standard CaC , MgC , or RbCI methods, protoplast fusion methods or transfection of naked or encapsulated nucleic acids using calcium phosphate precipitation, microinjection, viral infection, and electroporation.
  • a polynucleotide is derived from a mammal, such as a human.
  • Polynucleotides can also be synthesized in the laboratory, for example, using an automatic synthesizer.
  • An amplification method such as PCR can be used to amplify polynucleotides from either genomic DNA or cDNA encoding the polypeptides.
  • Polynucleotide molecules encoding a variant polypeptide can also be isolated by a gene amplification method such as PCR using a portion of a nucleic acid molecule DNA encoding a polypeptide shown in SEQ ID NOs: 60-65, 67-74, 88- 99 or fragments thereof as the probe.
  • Polynucleotides and fragments thereof can be used, for example, as probes or primers to detect the presence of SAS1 B polynucleotides in a sample, such as a biological sample.
  • a biological sample can be, e.g., lymph node or tissue aspirate, serum, lymphocytes, whole blood, cellular suspension, ovarian cyst fluid, pap smear, plasma, circulating tumor cells, tumor cells or tissue, ascites fluid, urine, or fluid effusion.
  • the ability of such probes to specifically hybridize to polynucleotide sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • Polynucleotide probes can hybridize to complementary sequences in a sample such as a biological sample, for example, lymph tissue.
  • Polynucleotides from the sample can be, for example, subjected to gel electrophoresis or other size separation techniques or can be dot blotted without size separation.
  • the polynucleotide probes can be labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or by kinase, biotin, fluorescent probes, and chemiluminescent probes.
  • the polynucleotides from the sample are then treated with the probe under hybridization conditions of suitable stringencies.
  • the stringency of hybridization conditions for a polynucleotide encoding a variant polypeptide to a polynucleotide encoding polypeptides shown in SEQ I D NOs: 1 -51 , 53-59, 66, 75-87, 100-1 1 1 can be, for example, 10% formamide, 5 x SSPE, 1 x Denhart's solution, and 1 x salmon sperm DNA (low stringency conditions). Such conditions include 25% formamide, 5 x SSPE, 1 x Denhart's solution, and 1 x salmon sperm DNA (moderate stringency conditions), including 50% formamide, 5 x SSPE, 1 x Denhart's solution, and 1 x salmon sperm DNA (high stringency conditions).
  • an isolated polynucleotide is a nucleic acid molecule that is not immediately contiguous with one or both of the 5' and 3' flanking sequences with which it is normally contiguous when present in a naturally occurring genome. Therefore, an isolated polynucleotide can be, for example, a polynucleotide that is incorporated into a vector, such as a plasmid or viral vector, a polynucleotide that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that where it naturally occurs); and a polynucleotide that exists as a separate molecule such as a polynucleotide produced by PCR amplification, chemically synthesis, restriction enzyme digestion, or in vitro transcription.
  • a vector such as a plasmid or viral vector
  • a polynucleotide that is incorporated into the genome of a heterologous cell or the genome of a homologous
  • An isolated polynucleotide is also a nucleic acid molecule, such as a recombinant nucleic acid molecule that forms part of hybrid polynucleotide encoding additional polypeptide sequences that can be used for example, in the production of a fusion protein.
  • a polynucleotide can also comprise one or more expression control sequences such as promoters or enhancers, for example.
  • a polynucleotide can be present in a vector, such as, for example, an expression vector. If desired, polynucleotides can be cloned into an expression vector comprising, for example, promoters, enhancers, or other expression control sequences that drive expression of the polynucleotides in host cells. The polynucleotides can be operably linked to the expression control sequences.
  • One embodiment provides methods of detecting SAS1 B polypeptides in a sample.
  • the methods comprise contacting the sample suspected of containing SAS1 B polypeptides with an antibody or antigen binding portion thereof (e.g., antibodies shown in Table 1 or antibodies and antigen binding portions thereof described herein) to form SAS1 B/antibody complexes.
  • an antibody or antigen binding portion thereof e.g., antibodies shown in Table 1 or antibodies and antigen binding portions thereof described herein
  • Another embodiment provides a method of detection of SAS1 B-positive cells
  • a test sample comprising contacting one or more antibodies or antigen-binding portions thereof (e.g., antibodies 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, 31 H1/31 K1 , antigen-binding fragments thereof, and antibody molecules that compete with 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1 /9K5, 31 H1/31 K1 for binding to SAS1 B) with the test sample under conditions that allow SAS I B-positive cell/antibody complexes to form.
  • the cells can be permeabilized or cell lysates.
  • the SAS1 B positive cell/antibody complexes are then detected.
  • the detection of SAS1 B positive cell/antibody complexes is an indication that SAS1 B cells are present in the test sample.
  • the test sample can be, e.g., lymph node or tissue aspirate, ovarian cyst fluid, pap smear, serum, whole blood, cellular suspension, lymphocytes, plasma, circulating tumor cells, tumor cells or tissue, ascites fluid, urine, or fluid effusion.
  • Polypeptide/antibody or SAS I B-positive cell/antibody complexes can be detected by any method known in the art, enzyme-linked immunosorbent assay (ELISA), multiplex fluorescent immunoassay (MFI or MFIA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoblotting analysis, an immunohistochemistry method, immunofluorescence assay, In situ hybridization, fluorescence-activated cell sorting (FACS) or a combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • MFI or MFIA multiplex fluorescent immunoassay
  • RIA radioimmunoassay
  • sandwich assay sandwich assay
  • western blotting immunoblotting analysis
  • an immunohistochemistry method immunofluorescence assay
  • FACS fluorescence-activated cell sorting
  • An immunoassay for SAS1 B can utilize one antibody or several different antibodies. Immunoassay protocols can be based upon, for example, competition, direct reaction, or sandwich type assays using, for example, labeled antibody. Antibodies can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope and bioluminescent labels.
  • Antibodies or antigen-binding portions thereof can be bound to a support and used to detect the presence of SAS1 B.
  • Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magletite.
  • Antibodies can be used in a method of the diagnosis of a hyperproliferative disorder by obtaining a test sample from, e.g., a human or animal suspected of having a hyperproliferative disorder.
  • the test sample is contacted with antibodies or antigen-binding portions thereof under conditions enabling the formation of antibody- antigen complexes (i.e., immunocomplexes).
  • antibody- antigen complexes i.e., immunocomplexes.
  • One of skill in the art is aware of conditions that enable and are appropriate for formation of antigen/antibody complexes.
  • the amount of antibody-antigen complexes can be determined by methodology known in the art.
  • a level that is higher than that formed in a control sample indicates the presence of a hyperproliferative disorder.
  • a control sample is a sample that does not comprise any SAS1 B polypeptides, SAS1 B-positive cells, or antibodies specific for SAS1 B.
  • the amount of antibody/antigen complexes or antibodies bound to SAS1 B-positive cells or cell lysates can be determined by methods known in the art.
  • a hyperproliferative disorder can be a neoplastic disorder (e.g., cancer, sarcoma, adenocarcinoma, adenocarcinoma of the lung, squamous carcinoma of the lung, malignant mixed mullerian tumor, lymphoma, breast cancer, ovarian cancer, colorectal cancer, liver cancer, uterine cancer, endometrial cancer, pancreatic cancer, lung cancer, etc.) or a hematologic malignancy (e.g., leukemia, etc.).
  • a hyperproliferative disorder can be detected in a subject.
  • a biological sample is obtained from the subject.
  • One or more antibodies or antigen-binding portions thereof are contacted with the biological sample under conditions that allow SAS1 B polypeptide/antibody complexes (including, for example, a complex of an antibody or antigen-binding portion thereof and a cell expressing SAS1 B on its surface) to form.
  • SAS1 B polypeptide/antibody complexes including, for example, a complex of an antibody or antigen-binding portion thereof and a cell expressing SAS1 B on its surface
  • the SAS1 B polypeptide/antibody complexes are detected.
  • the detection of the SAS1 B polypeptide/antibody complexes is an indication that the mammal has a hyperproliferative disorder.
  • the lack of detection of the polypeptide/antibody complexes is an indication that the mammal does not have a hyperproliferative disorder.
  • the SAS1 B polypeptide/antibody complex is detected when an indicator reagent, such as an enzyme conjugate, which is bound to the antibody, catalyzes a detectable reaction.
  • an indicator reagent comprising a signal generating compound can be applied to the polypeptide/antibody complex under conditions that allow formation of a polypeptide/antibody/indicator complex.
  • the polypeptide/antibody/indicator complex is detected.
  • the polypeptide or antibody can be labeled with an indicator reagent prior to the formation of a polypeptide/antibody complex.
  • the method can optionally comprise a positive or negative control.
  • one or more antibodies are attached to a solid phase or substrate.
  • a test sample potentially comprising a polypeptide of this disclosure is added to the substrate.
  • One or more antibodies that specifically bind SAS1 B are added.
  • the antibodies can be the same antibodies used on the solid phase or can be from a different source or species and can be linked to an indicator reagent, such as an enzyme conjugate. Wash steps can be performed prior to each addition.
  • a chromophore or enzyme substrate is added and color is allowed to develop. The color reaction is stopped and the color can be quantified using, for example, a spectrophotometer.
  • Assays include, but are not limited to those based on competition, direct reaction or sandwich-type assays, including, but not limited to enzyme linked immunosorbent assay (ELISA), multiplex fluorescent immunoassay (MFI or MFIA) western blot, I FA, radioimmunoassay (RIA), western blot, hemagglutination (HA), fluorescence polarization immunoassay (FPIA), in situ hybridization, fluorescence- activated cell sorting (FACS), and microtiter plate assays (any assay done in one or more wells of a microtiter plate).
  • ELISA enzyme linked immunosorbent assay
  • MFI or MFIA multiplex fluorescent immunoassay
  • I FA radioimmunoassay
  • RIA radioimmunoassay
  • FPIA fluorescence polarization immunoassay
  • FACS fluorescence- activated cell sorting
  • microtiter plate assays any assay done in one or more wells of
  • Assays can use solid phases or substrates or can be performed by immunoprecipitation or any other methods that do not utilize solid phases.
  • a solid phase or substrate is used, one or more antibodies or antigen-binding portions thereof are directly or indirectly attached to a solid support or a substrate such as a microtiter well, magnetic bead, non-magnetic bead, column, matrix, membrane, fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, such as, polyethylene, polypropylene, or polyester), sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature).
  • synthetic or natural fibers e.g., glass or cellulose-based materials or thermoplastic polymers, such as, polyethylene, polypropylene, or polyester
  • sintered structure composed of particulate materials
  • particulate materials e.g., glass
  • substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
  • suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • one or more antibodies or antigen-binding portions thereof can be coated on a solid phase or substrate.
  • a test sample suspected of containing SAS1 B polypeptides or SAS I B-positive cells is incubated with an indicator reagent comprising a signal generating compound conjugated to an antibody or antigen-binding antibody fragment specific for SAS1 B (indicator reagent composition) for a time and under conditions sufficient to form antigen/antibody complexes of either SAS1 B polypeptides of the test sample to the antibodies or antigen-binding fragments thereof of the solid phase or the indicator reagent compound.
  • the reduction in binding of the indicator reagent can be quantitatively measured.
  • a measurable reduction in the signal compared to the signal generated from a confirmed negative SAS1 B test sample indicates the presence of SAS1 B in the test sample.
  • This type of assay can quantitate the amount of SAS1 B in a test sample.
  • polypeptide/antibody complex or a polypeptide/antibody/indicator complex can be detected by e.g. , radiometric, colorimetric, fluorometric, size-separation, or precipitation methods.
  • detection of a polypeptide/antibody complex is by the addition of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound.
  • Indicator reagents comprising signal generating compounds (labels) associated with a polypeptide/antibody complex can be detected using the methods described above and include chromogenic agents, catalysts such as enzyme conjugates fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like.
  • enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta- galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • Formation of the complex (including, for example, antibodies 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, 31 H1/31 K1 , antigen-binding fragments thereof, and antibody molecules that compete with 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1 /9K5, 31 H1/31 K1 for binding to SAS1 B) can be indicative of the presence of SAS1 B-positive cells in a test sample. Therefore, the methods can be used to diagnose a hyperproliferative disease in a mammal.
  • the methods can also indicate the amount or quantity of SAS1 B in a test sample.
  • the amount of SAS1 B present is proportional to the signal generated.
  • it can be diluted with a suitable buffer reagent, concentrated, or contacted with a solid phase without any manipulation. For example, it usually is preferred to test samples that previously have been diluted, or concentrated specimens, in order to determine the presence and/or amount of SAS1 B present.
  • a polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present as when the polypeptide is expressed in a native cell, or in systems that result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.
  • systems e.g., cultured cells, which result in substantially the same post-translational modifications present as when the polypeptide is expressed in a native cell, or in systems that result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.
  • An expression vector can be, for example, a plasmid, such as pBR322, pUC, or ColE1 , or an adenovirus vector, such as an adenovirus Type 2 vector or Type 5 vector.
  • Vectors suitable for use include, for example, bacterial vectors, mammalian vectors, viral vectors (such as retroviral, adenoviral, adeno-associated viral, herpes virus, simian virus 40 (SV40)) and baculovirus-derived vectors for use in insect cells.
  • Polynucleotides in such vectors can be operably linked to a promoter, which is selected based on, e.g., the cell type in which expression is sought.
  • the expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody.
  • An embodiment includes host cells containing polynucleotides encoding an antibody (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody, or a fragment or variant thereof), operably linked to a heterologous promoter.
  • an antibody e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody, or a fragment or variant thereof
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule.
  • Host cells into which vectors, such as expression vectors, comprising polynucleotides can be introduced include, for example, prokaryotic cells (e.g., bacterial cells) and eukaryotic cells (e.g., yeast cells; fungal cells; plant cells; insect cells; and mammalian cells). Such host cells are available from a number of different sources that are known to those skilled in the art, e.g., the American Type Culture Collection (ATCC), Manassas, VA. Host cells into which the polynucleotides have been introduced, as well as their progeny, even if not identical to the parental cells, due to mutations, are included herein. Host cells can be transformed with the expression vectors to express the antibodies or antigen-binding fragments thereof. Host cells expressing antibodies or antigen-binding fragments thereof include cells and hybridomas transformed with a polynucleotide described herein.
  • prokaryotic cells e.g., bacterial cells
  • One embodiment provides methods of producing a recombinant cell that expresses an SAS1 B antibody, antigen-binding fragment thereof or portion thereof, comprising transfecting a cell with a vector comprising a polynucleotide.
  • An SAS1 B antibody, or fragment, or portion thereof can then be produced by expressing the polypeptide in the recombinant host cell.
  • Isolation and purification of polypeptides produced in the systems described above can be carried out using conventional methods, appropriate for the particular system. For example, preparative chromatography and immunological separations employing antibodies, such as monoclonal or polyclonal antibodies, can be used.
  • Antibodies and antigen-binding fragments thereof e.g., antibodies 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, 31 H1/31 K1 , antigen-binding fragments thereof, and antibody molecules that compete 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1 /7K3, 9H1/9K5, 31 H1/31 K1 for binding to SAS1 B) that specifically bind SAS1 B can be conjugated to a therapeutic agent or effector molecule to form an "antibody-drug conjugate".
  • a therapeutic agent is an agent with a biological activity directed against a particular target molecule or a cell bearing a target molecule.
  • Therapeutic agents can include, for example, various drugs such as vinblastine, daunomycin, cytotoxins such as maytansinoids and maytansinoid analogs, a prodrug, tomaymycin derivatives, taxoids, a leptomycin derivative, CC- 1065 and CC-1065 analogs, encapsulating agents (such as liposomes) that contain pharmacological compositions, therapeutic agents, toxins (e.g. , ricin, abrin, diphtheria toxin and subunits thereof, botulinum toxins A through F, variants of toxins (see, e.g., U.S. Pat.
  • Pseudomonas exotoxin PE
  • Pseudomonas exotoxin PE
  • U.S. Pat. No. 5,602,095 Pseudomonas exotoxin
  • variants thereof see, e.g. U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821 ,238; and 5,854,044
  • PCT Publication No. WO 99/51643 Pai et al., Proc. Natl. Acad. Sci. USA 88:3358, 1991 ; Kondo et al., J. Biol. Chem. 263:9470, 1988; Pastan et al., Biochim.
  • radioactive agents such as 125 l, 32 P, 14 C, 3 H and 35 S and other labels, target moieties and ligands.
  • An effector molecule is a small molecule that selectively binds to a protein and regulates its biological activity.
  • Effector or therapeutic molecules can be linked to an antibody using any number of means known to those of skill in the art, for example by covalent or noncovalent attachment.
  • Therapeutic agents or effector molecules that are polypeptides will typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule or therapeutic agent.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of known linker molecules.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule or therapeutic agent.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • antibody-drug conjugates can comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule or therapeutic molecule from the antibody can be accomplished by, for example, enzymatic activity or conditions to which the antibody-drug conjugate is subjected either inside the target site or in the vicinity of the target site.
  • Antibodies can be labeled with a detectable moiety.
  • Detectable moieties include, for example, fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1 -napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, bioluminescent markers (e.g., luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP)), enzymes (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase, glucose oxidase), a magnetic agent (e.g.
  • Detectable moieties can be attached to antibodies by spacer arms of various lengths to reduce potential steric hindrance.
  • Radioimmunoconjugates allow radiation therapy to be delivered directly to the surface of tumor cells in relatively high doses while sparing normal tissues from the effects of radiation. Radioimmunoconjugates can inhibit tumor cell proliferation and microvascularization around tumor cells and can be used to eradicate the last microscopic clusters of tumor cells after treatment.
  • An antibody of the invention can be conjugated to an alpha emitter or a beta emitter, for example 177 Lu, 131 l, 90 Y, 188 Re, 186 Re, 67 Cu, 212 Bi, 213 Bi or 21 1 At, 149 Tb, 225 Ac, 212 Pb, 225 Ac, 227 Th, 199 Ah, 199 Au, 194 lr, 166 Ho, 159 Gd, 153 Sm, 149 Pm, 142 Pr, 111 Ag, 109 Pd, and 77 As.
  • an alpha emitter or a beta emitter for example 177 Lu, 131 l, 90 Y, 188 Re, 186 Re, 67 Cu, 212 Bi, 213 Bi or 21 1 At, 149 Tb, 225 Ac, 212 Pb, 225 Ac, 227 Th, 199 Ah, 199 Au, 194 lr, 166 Ho, 159 Gd, 153 Sm, 149 Pm, 142 Pr, 111 Ag, 109 Pd,
  • a radioimmunoconjugate can be comprised of an antibody or an antigen- binding fragment of an antibody that specifically binds SAS1 B linked to radionuclide molecule via a linker.
  • Any type of linker with sufficient complexing ability and a functional group allowing direct or indirect conjugation to a protein or a peptide can be used.
  • an antibody or binding fragment thereof can be linked to a radionuclide by a chelating agent.
  • a chelating agent is attached to the antibody or binding fragment thereof and functions to chelate the radionuclide.
  • the binding or linking of the radionuclide to the antibody can be done in any manner known in the art, as long as the targeting specificity of the antibody or binding fragment thereof is not decreased by a substantial amount.
  • Chelating agents are known in the art and include, for example, DOTA, cDTPA, TETA and CHX-A-DTPA (cyclohexyldiethylenetriamine pentaacetic acid).
  • a radionuclide can be attached to an antibody or binding fragment thereof by reacting a bifunctional chelating agent, e.g., p-SCN-bn-DOTA with the antibody, followed by purification to remove unconjugated chelator, and then reaction of the chelator antibody conjugate with the radionuclide, followed by purification to remove any unconjugated radionuclide.
  • a bifunctional chelating agent e.g., p-SCN-bn-DOTA
  • the chelation agent and the radionuclide can be combined and then subsequently conjugated to the antibody.
  • bifunctional cyclic chelating agents examples include p-SCN-bn-DOTA, and DOTA-NHS-ester.
  • Bifunctional linear chelators like p-SCN-Bn-DTPA and CHX-A"-DTPA can also be used.
  • Cyclic, linear or branched chelating agents can be used, for example polyaminopolyacid chelating agents that comprise a linear, cyclic or branched polyazaalkane backbone with acidic (e.g. carboxyalkyl) groups attached at backbone nitrogens.
  • Suitable chelating agents include DOTA derivatives such as p-isothiocyanatobenzyl-1 ,4, 7, 10-tetraazacyclododecane-1 , 4, 7, 10-tetraacetic acid (p-SCN-Bz-DOTA) and DTPA derivatives such as p-isothiocyanatobenzyl- diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA).
  • DOTA derivatives such as p-isothiocyanatobenzyl-1 ,4, 7, 10-tetraazacyclododecane-1 , 4, 7, 10-tetraacetic acid (p-SCN-Bz-DOTA)
  • DTPA derivatives such as p-isothiocyanatobenzyl- diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA).
  • An embodiment provides a pharmaceutical composition
  • a pharmaceutical composition comprising a radioimmunoconjugate as described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • Acceptable pharmaceutical carriers include but are not limited to non-toxic buffers, fillers, isotonic solutions, and the like.
  • the carrier can contain a radiolytic stabilizer, e.g., ascorbic acid, which can protect the integrity of the radioimmunoconjugate during storage and shipment.
  • a radioimmunoconjugate can be provided as a pharmaceutical composition comprising a radionuclide linked to an antibody or a fragment thereof dissolved in a buffer solution, which substantially maintains the chemical integrity of the radioimmunoconjugate.
  • the pharmaceutical composition can be physiologically acceptable for infusion into patients.
  • a patient can be treated by administering one or more radioimmunoconjugates to a patient.
  • the one or more radioimmunoconjugates can be administered to a patient in conjunction with one or more clearing agents or chelating agents to prevent unbound radioisotope from accumulating in and damaging organs and tissues in the body.
  • a pharmaceutical composition of the invention can by administered by, for example, intravenous infusion or intravenous injection.
  • a pharmaceutical composition can be administered directly in a vein by a peripheral cannula connected to a drip chamber that prevents air embolism and allows an estimate of flow rate into the patient.
  • a pharmaceutical composition can be administered in a repeated fashion (e.g., 2, 3, 4, 5, 6, or more times over, e.g. , 1 , 2, 3, 4, 5, days or more, or 1 , 2 3, 4, 5 weeks or more, or 1 , 2, 3, 4, 5, weeks or more).
  • a radioimmunoconjugate can be administered in a repeated fashion, but with different radionuclides, e.g., beta-radioimmunotherapy can be followed by alpha-radioimmunotherapy or vice versa.
  • a radioimmunoconjugate can be administered alone or in combination with or in addition to one or more additional therapies.
  • An additional therapy can be, for example, pretreatment, chemotherapy, monoclonal antibody therapy, surgery, radioimmunotherapy, photodynamic therapy, bone marrow transplantation, and stem cell transplantation and/or therapy.
  • the antibody dosing is about 1 , 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more mg per patient, and a radionuclide amounting to about 1 , 2, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200 or more MBq/kg of bodyweight.
  • kits for the production of a radioimmunoconjugate which can comprise two or more vials, wherein one vial contains a conjugate comprising a linker such as a chelator linked to an antibody or antibody fragment of the disclosure and a second vial contains a radionuclide. Some procedures can be performed, e.g., radiolabeling and/or purification before infusion. The contents of one or more of the vials can be dried, lyophilized, or in a solution. In an embodiment the radioimmunoconjugate is produced by mixing the content of the two vials.
  • Antibodies and antigen-binding fragments thereof e.g., antibodies 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, 31 H1/31 K1 , antigen-binding fragments thereof, and antibody molecules that compete with 7H2, 6C1 , 1 H2/1 K2, 1 H3/1 K3, 2H2/2K5, 6B1 , 3F2, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, 31 H1/31 K1 for binding to SAS1 B) can be conjugated to a cytotoxic agent, such as duocarmycin, maytansanoids, and auristatins, to form a drug having specific cytotoxicity towards SAS I B-expressing cells by targeting the drug to SAS1 B.
  • Cytotoxic conjugates comprising such antibodies and/or antigen-binding fragments thereof and a drug or cytotoxin can be used as a therapeutic for treatment of hyperproliferative disorders such as such as any SAS I B-positive cancer, including, but not limited to, uterine cancer, pancreatic cancer, and ovarian cancer.
  • One embodiment provides methods of treating or preventing hyperproliferative disorders comprising administering an effective amount of an antibody or antigen-binding fragment thereof or an antibody-drug conjugate to a mammal in need thereof.
  • An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, an effective amount is one that achieves the desired therapeutic effect.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • An effective amount of a pharmaceutical composition i.e. , an effective dosage
  • the compositions can be administered from one or more times per day to one or more times per week; including once every other day.
  • treatment of a subject with an effective amount of the pharmaceutical compositions described herein can include a single treatment or a series of treatments.
  • Treatment means the administration of one or more pharmaceutical compositions to a subject.
  • treatment also includes an adjustment (e.g. , increase or decrease) in the dose or frequency of one or more pharmaceutical agents that a subject can be taking, the administration of one or more new pharmaceutical agents to the subject, or the removal of one or more pharmaceutical agents from the subject's treatment plan.
  • Treatment also refers to any amelioration of one or more symptoms of a hyperproliferative disease, improvement in patient survival, or the reversal of the disease.
  • a subject can be an animal, for example, a mammal, a human, monkey, dog, cat, horse, cow, pig, goat, rabbit, or mouse.
  • a “pharmaceutical composition” is a sterile or aseptic composition of an antibody or antigen-binding fragment thereof or antibody-drug conjugate formulated with a pharmaceutically acceptable carrier, which can be safely administered to a subject.
  • the pharmaceutical composition does not cause undesirable side effects when administered to a patient that outweigh the beneficial effects.
  • One embodiment provides a pharmaceutical composition for the treatment of a hyperproliferative disorder in a mammal, which comprises an effective amount of an antibody or antigen-binding portion thereof or an antibody-drug conjugate and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be used for the treatment of cancer, including (but not limited to) the following: carcinoma, including that of the bladder, breast, endometrium, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibro
  • “Pharmaceutically-acceptable carriers” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.
  • suitable carriers, diluents and/or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combination thereof.
  • Isotonic agents, such as sugars, polyalcohols, or sodium chloride can be present in compositions.
  • Suitable carriers include, for example: Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serum albumin; 0.9% saline (0.9% w/v sodium chloride (NaCI)), and 5% (w/v) dextrose. Carriers can also contain an antioxidant such as tryptamine and a stabilizing agent such as TWEEN20® (polysorbate).
  • Administration can be by any method, including, for example parenteral (e.g. intravenous, intramuscular, intraperinoneal, subcutaneous, intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional).
  • parenteral e.g. intravenous, intramuscular, intraperinoneal, subcutaneous, intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional.
  • Compositions can be administered intravenously as a bolus or by continuous infusion over a period of time.
  • Sterile or aseptic compositions for parenteral administration can be prepared by incorporating the antibody, antigen-binding fragment or antibody-drug conjugate in the required amount in the appropriate solvent, followed by sterilization by microfiltration.
  • solvent or vehicle include, for example, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as a combination thereof.
  • Isotonic agents such as sugars, polyalcohols, or sodium chloride can be present in compositions. These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents.
  • Sterile compositions for parenteral administration can also be prepared in the form of sterile solid compositions, which can be dissolved at the time of use in sterile water or any other injectable sterile medium.
  • the antibodies, antigen-binding fragments thereof or antibody-drug conjugates can also be orally administered as a solid composition (tablets, pills, powders gelatin capsules, sachets) or granules) or liquid compositions (pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil).
  • These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing products.
  • the doses depend on the desired effect, the duration of the treatment and the route of administration used; they are generally between 5 mg and 1000 mg per day orally for an adult with unit doses ranging from 1 mg to 250 mg of active substance.
  • Antibodies, antibody-binding portions thereof, or antibody-drug conjugates can be used for the treatment of a hyperproliferative disorder in a mammal.
  • the antibodies, antibody-binding portions thereof, or antibody-drug conjugates can also be used to treat the neovascularization of a tumor.
  • an embodiment provides a method for inhibiting the growth of selected cell populations comprising contacting target cells, or tissue containing target cells, with an effective amount of an antibody, antigen-binding fragment or antibody-drug conjugate, or an antibody, antigen-binding fragment or a therapeutic agent comprising a cytotoxic conjugate, either alone or in combination with other cytotoxic or therapeutic agents.
  • SAS1 B can be practiced in vitro, in vivo, or ex vivo.
  • “Inhibiting growth” means slowing the growth of a cell, decreasing cell viability, causing the death of a cell, lysing a cell and inducing cell death, over a short period of time (e.g., minutes to hours) or a long period of time (e.g. , days to months).
  • Examples of in vitro uses include treatments of autologous bone marrow prior to their transplant into the same patient in order to kill diseased or malignant cells; treatments of bone marrow prior to its transplantation in order to kill competent T cells and prevent graft-versus-host-disease (GVHD); treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.
  • treatments of autologous bone marrow prior to their transplant into the same patient in order to kill diseased or malignant cells
  • treatments of bone marrow prior to its transplantation in order to kill competent T cells and prevent graft-versus-host-disease (GVHD)
  • treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.
  • Examples of clinical ex vivo use include the removal of tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from autologous or allogeneic bone marrow or tissue prior to transplant in order to prevent graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the antibody, the antigen-binding fragment, or the antibody-drug conjugate can be supplied as solutions that are sterile and contain appropriate levels of endotoxin.
  • suitable protocols of antibody-drug conjugate administration are as follows.
  • Antibodies, antigen-binding fragments thereof or antibody-drug conjugates can be given weekly for 4 weeks as an i.v. bolus each week.
  • Bolus doses are given in 50 to 100 ml of normal saline to which 5 to 10 ml of human serum albumin can be added. Dosages can be 10 ⁇ g to 100 mg per administration, i.v. (range of 100 ng to 1 mg/kg per day). Dosages can range from 50 ⁇ g to 30 mg.
  • Dosages can range from 1 mg to 20 mg. After four weeks of treatment, the patient can continue to receive treatment on a weekly basis. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc. , can be determined by one of ordinary skill in the art as the clinical situation warrants.
  • the antibodies or antigen-binding fragments can also be used to detect
  • SAS1 B in a biological sample in vitro or in vivo can be used to determine the level of SAS1 B in a tissue or in cells derived from the tissue.
  • the tissue can be diseased tissue, a tumor or a biopsy of a tumor.
  • the tissue or biopsy thereof can be frozen, fixed, permeabilized or non-permeabilized.
  • the above-described method can be used to diagnose a cancer in a subject known to or suspected to have a cancer, wherein the level of SAS1 B measured in said patient's tissues, blood or serum is compared with that of a normal reference subject or standard. The method can then be used to determine whether a tumor or cells of tissue of the patient expresses SAS1 B, which may suggest that the tumor or patient will respond well to treatment with the antibodies, antigen-binding fragments or antibody-drug conjugates described herein.
  • a method for diagnosis is also provided in which labeled antibodies, antigen- binding fragments thereof, or antibody-drug conjugates are administered to a subject suspected of having a cancer, and the distribution of the label within the body of the subject is measured or monitored.
  • the SAS1 B protein is encoded by the Astl gene (astacin-like metalloendopeptidase).
  • Astl gene astacin-like metalloendopeptidase
  • ASTL expression is upregulated in lung adenocarcinomas.
  • ASTL has a gain of copy number ranking of 1389, placing ASTL in the top 8% of genes upregulated in lung adenocarcinomas. This data in primary tumors is supported by upregulation of ASTL in lung adenosquamous and adenocarcinoma cell lines.
  • ASTL expression in several squamous and adenocarcinoma lung cancer cell lines has been confirmed.
  • PCR amplification of the human SAS1 B catalytic domain of 579 bp occurred in lung cancer cell lines NCI-H226, A549, HEK-293, human ovary, MMMT539, while the control water was negative.
  • Western blotting of protein extracts from lung cancer H226 and A549 cell lines using a guinea pig polyclonal anti-human SAS1 B antibody showed expression of full length (46 kD) and truncated SAS1 B proteins in both lung cancer cell lines H226 and A549.
  • SAS1 B can be localized to the surfaces of both H226 and A549 cells.
  • the human SAS1 B protein is encoded by the Astl gene (astacin-like metalloendopeptidase).
  • Astl gene astacin-like metalloendopeptidase
  • ASTL expression is upregulated in lung adenocarcinomas.
  • ASTL has a gain of copy number ranking of 1389, placing ASTL in the top 8% of genes upregulated in lung adenocarcinomas.
  • This data in primary tumors is supported by upregulation of ASTL in lung adenosquamous and adenocarcinoma cell lines.
  • ASTL expression was confirmed in several squamous and adenocarcinoma lung cancer cell lines.
  • SAS1 B A diagram of the human SAS1 B polypeptide is shown in Figure 1 .
  • SAS1 B has been divided into domains. These include a classic signal peptide, propetide, putative transmembrane, catalytic region embedded in overall larger protease domain and C-terminus domain.
  • SAS1 B shows protein microheterogeneity in both mice and humans.
  • SAS1 B has several splice variant forms: SV-A, SV-B, SV-C, SV-D, SV- E, and SV-F.
  • Splice variants A and C were PCR amplified using variant specific forward and reverse primers.
  • SV-A and SV-C were amplified from the following RNA sources: 1 ) SNU539 cell line (malignant mixed Mullerian tumor); 2) pancreatic ductal adenocarcinoma (PDAC) mouse xenograft tumor samples (human primary patient tumors were affixed into pancreas of nude mice and then propagated; tumor RNA interrogated here from approximately F5 generation); 3) human primary patient head and neck squamous cell carcinoma (HNSCC) samples.
  • PCR products were subcloned and sequenced and sequence identity was used to confirm SV-A, SV-C, or SV-D splice variants.
  • Splice variant B was identified by sequencing data generated by subcloning PCR products from SV-A primer set. DNA sequences were identified from all three RNA sources listed above confirming presence of SV- B transcript in different tumor types.
  • Splice variant D was PCR amplified using variant specific primers from SNU539 cell line RNA. Identification was confirmed by subcloning and sequencing PCR amplicon.
  • SV-B lacks 18 amino acids from Exon 8
  • SV-C has a novel N-terminal transmembrane domain
  • SV-D has a novel transmembrane domain and an ORF stop after Exon 7.
  • SV-E and SV-F have a novel transmembrane domain and an N- terminal ORF stop after Exon 4.
  • SV-E and SV-F have a C-terminal ORF that starts from extended Exon 5.
  • E. coli E. coli, and used to generate mouse antibodies.
  • the immunogen was denatured prior to generation of antibodies.
  • Monoclonal antibodies 6B1 and 3F2 were generated.
  • Amino acids 280-430 of splice variant A of human SAS1 B (“recombinant SAS1 B-CT”) were expressed in E. coli and used to generate mouse antibodies. Monoclonal antibodies 6C1 and 7H2 were generated. Clones 6C1 and 7H2 recognized recombinant SAS1 B-CT in a western blot demonstrating that they are positive clones.
  • Amino acids 280-430 (“recombinant SAS1 B-CT”) of splice variant A of human SAS1 B were expressed in E. coli and used to generate rabbit antibodies.
  • Monoclonal antibodies 1 H2/1 K2, 1 H3/1 K2, 2H2/2K5, 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1/7K3, 9H1/9K5, and 31 H1/31 K1 were generated.
  • Monoclonal antibodies 6C1 , 7H2, and 3A7 were screened by western blotting using SAS1 B-SVA transfected HEK293 T cell extracts.
  • HEK293 cells were transfected with SAS1 B-SVA plasmids to express the SAS1 B protein.
  • the cell lysate from the untransfected and transfected cells were prepared, electrophoresed, and blotted. Blots were then probed with the anti-SAS1 B-CT monoclonal antibodies (6C1 and 7H2), the negative control 3A7 antibody, SB3, which is a mouse monoclonal antibody known to react with SAS1 B-transfected cells, and an anti-His antibody.
  • SAS1 B-CT monoclonal antibodies 6C1 and 7H3 reacted with the 52 kDa SAS1 B protein.
  • 3A7 did not react with the SAS1 B protein.
  • a western blot analysis of the SAS1 B-anti-mouse and anti-rabbit monoclonal antibodies on endogenous SAS1 B using a colon cancer cell line SW480 cell lysate was performed.
  • the SW480 cell lysate was made in Celis extraction buffer.
  • a rabbit polyclonal antibody recognized a 65 kDa band and a higher molecular weight band of about ⁇ 120 kDa which was also recognized by rabbit 1 H3/1 K2 and mouse monoclonal 6B1 .
  • Mouse monoclonal antibody 3F2 recognized a strong band at ⁇ 100 kDa and 75 kDa and lower molecular weight bands at 37 kDa and 30 kDa.
  • An SAS1 B rabbit polyclonal antibody recognized mainly a ⁇ 65kDa band, a -50 kDa band (pi 4.5) and a ⁇ 37kDa (pi 7) band on a 2D Western blot in which the lysate was prepared in Celis.
  • the pattern shown by the rabbit monoclonal antibody 1 H3/1 K2 is similar to the one displayed on 1 D Western, showing multiple charge variants at around 120 kDa and pi 6.5.
  • 6B1 recognized a strong band at around 75 kDa at a pl ⁇ 5.5.
  • Mouse monoclonal 3F2 recognized a band little higher, very close to the spots recognized by rabbit monoclonal 1 H3 /1 K2, with slight acidic pi.
  • Mouse monoclonal 3A7 did not recognize the recombinant SAS1 B but recognized the E. coli protein contaminant in the immunogen. Therefore, it was used as a negative control. 3A7 did not recognize any major bands in a SNU539 cell lysate.
  • Both the eluted protein and the bead pellet was checked by western blotting for the presence of the SAS1 B protein using an anti-His antibody. Immmunoprecipitation from both 6C1 and 7H2 showed that both antibodies are capable of binding and capturing the recombinant SAS1 B.
  • Immunohistochemical experiments were performed with mouse ovarian tissue sections. Culture supernatants from 6B1 and 3F2 showed oocyte staining in Bouins fixed mouse ovarian section. The antibodies also showed immunoreactivity in oocytes within secondary antral follicles, while immunoreactivity was not seen in oocytes contained within primordial or primary follicles. Control mouse IgG showed no staining to any cell type.
  • Protein A purified monoclonal antibody 3F2 was used on either zinc formalin or neutral buffered formalin fixed monkey ovary section. Intense immunoreactivity to oocytes housed in secondary follicles was revealed. Oocytes in primordial and primary follicles were SAS1 B negative. Control mouse IgG did not stain any cell type.
  • Protein A purified monoclonal antibody 3F2 was used on neutral buffered formalin-fixed monkey normal breast/mammary glands, uterus, pancreas and kidney sections. The results revealed no immunoreactivity to any cell types. Control mouse IgG (Ms IgG) did not stain any cell type. SNU539 uterine cancer cells were injected subcutaneously into nude m ice. The cells formed solid tumors. Excised tumors were fixed in Bouin's fixative followed by SAS 1 B immunohistochem ical staining. Monoclonal antibody culture supernatants 6B 1 and 3F2 were used to stain xenograft tissue sections. Both antibodies immunostained the tumor section. Control mouse IgG (Ms IgG) did not stain any cells.
  • Monoclonal antibody culture supernatants 6B 1 and 3F2 were used to stain live SNU539 uterine cancer cells.
  • the antibodies did not immunoreact to mem brane structures indicating that the epitope for these antibodies could be buried within the cell.
  • the same antibodies when used to probe a western blot of the same cell line total protein extract, showed immunoreactivity to several protein bands in the native extract and also reacted to the recombinant HS4 antigen at ⁇ 35 kDa.
  • Control mouse IgG showed no immunostaining to the cells or extracts.
  • Zinc formalin-fixed clear cell papillary renal cell carcinoma cells were immunostained with monoclonal antibody 3F2. The antibody immunostained the tumor section. Control mouse IgG (Ms IgG) did not stain any cells.
  • FACS analysis was performed using SAS1 B positive cell lines and rabbit monoclonal antibodies. The following samples were used: un-stained cells, secondary antibody alone, positive Integrin oc4 Rb mAb, mAb 34 mock control, mAb 1 , mAb 2, mAb 3 (1 H2/1 K2), mAb 5 (1 H3/1 K2), and mAb 7 (2H2/2K5). 2 m illion cells/tube of SNU539 cells were allowed to recover for 2-3 hours at 37°C in media. Cells were blocked with warm 5% NGS media and chilled in 0.1 % azide containing media on ice.
  • the cells were then incubated with the rabbit monoclonal antibody supernatants mAb (8ug/m l) or controls in blocking media for 2 hours on ice.
  • the cells were washed with azide buffer.
  • the secondary antibody was goat anti-rabbit R-PE (R-phycoerythrin).
  • the cells were fixed with 4% PFA (paraformaldehyde) for 1 5 m ins at room temperature, washed and analyzed on the flow cytometer (Calibur).
  • the integrin oc4 monoclonal antibody was used as a positive control for the flow cytometry assay to determ ine expression of surface integrin protein on the cancer cells.
  • the mock control is the media in which the monoclonal antibodies were obtained. These were SAS 1 B negative.
  • Monoclonal antibodies 1 , 3 and 5 showed good surface staining by flow cytometry.
  • H EK293T cells were seeded at ⁇ 1 00, 000 per well of 24 well plate containing a lysine-coated 12 m m coverslip. After 24 hours the cells were transfected with 1 ug of SV-A plasm id with 2 ul of TU RBOFECT ® transfection reagent followed by no change of media. 48 h later the cells were PFA fixed and methanol permeabilized for an IF study as follows.
  • the cells were removed from cell culture media and fixed in 4% PFA in PBS, 300 ul for 1 5 m in.
  • the cells were washed in 1 m l PBS in wells for 3 times and permeabilized in methanol (1 00%) for 1 5 m inutes in 0.5 m l at room temperature.
  • the cells were washed in PBS (1 m l) 3 times and blocked in complete culture media (DM EM) with 5% normal goat serum (NGS) for 30 m in (i.e. , BB, blocking buffer).
  • the first antibody in BB was added for 1 h at 1 0 ug/m l for monoclonal antibodies 3F2 and 6B 1 and 5 ug/m l for anti-V5 DYLIGHT® 488 rabbit (a rabbit polyclonal antibody to V5 tag).
  • the cells were washed in PBS 3 times.
  • the secondary antibody in BB (goat anti-mouse ALEXA FLUOR® 594) was added for 1 h at 5 ug/m l (1 :200 dilution).
  • the cells were washed in PBS 3 times.
  • the cells were mounted in SLOWFADE® antifade mountant with DAP I nuclear stain ((3ul) and stored at 4°C before confocal imaging.
  • the mock transfected H EK293T cells showed no V5 signal or SAS 1 B signal when probed with anti-V5 antibody or mouse IgG control antibody or secondary antibody alone.
  • the anti-V5 antibody confirmed expression of human SAS1 B with C-terminal V5 tag in fixed and permeabilized HEK293T cells.
  • the V5 tag antibody confirmed the expression of SAS1 B SV-A epitopes in transfected HEK293T cells.
  • the V5 tag antibody confirmed the expression of SAS1 B SV-A epitopes in transfected HEK293T cells.
  • SAS1 B mAbs 3F2 and 6B1 IF colocalizes (merge) with the V5-tag epitope signal confirming the specificity of 3F2 and 6B1 monoclonal antibodies for the SAS1 B epitope.
  • Anti-Human SAS1 B mouse monoclonal antibodies reactive to a SAS1 B epitope on live cancer cell surfaces were identified by indirect immuno- fluorescence (IF) for the development of therapeutic antibodies.
  • M1 (renal cancer cell line) and MMMT539 (uterine cancer cell line) cells were seeded at 20,000 cells in 1 ml per well on collagen coated coverslips in 24 well plates. The media was replaced 22 h post seeding. For antibody probing, cells were kept on ice for ⁇ 8 to 18 min following 47 h post seeding.
  • the first antibody was added at 50 ug or 20 ug or 10 ug/ml in complete culture media with 15 mM NaN3 (pre-chilled) incubated on ice for 60 min, and the cells washed in complete culture media with 15 mM NaN3 in cold 1 ml, x3.
  • the second antibody, goat-antimouse-AF488 (pre-chilled) was added to the wells at 5ug/ml in complete culture media with 15 mM NaN3 for 60 min on ice. The cells were washed in complete media with 15 mM NaN3, 1 ml, x3 on ice.
  • EGFR Epidermal growth factor receptor
  • EGFR is a transmembrane glycoprotein and member of protein kinase superfamily. Both fluorescence and confocal microscopy confirmed the EGFR expression on the cell surface of the live M1 renal carcinoma cell line. Most of the cells expressed the receptor and validated the live cell surface IF staining methodology for the screening of anti-human SAS1 B mouse monoclonal antibodies.
  • the receptor molecule was found on the cell surfaces.
  • Anti-human CABYR monoclonal antibody 3A4 was used as a negative control for live MMMT539 cells.
  • CABYR is a calcium binding cytoskeleton sperm protein with no transmembrane domain.
  • Anti-CABYR mAb 3A4 showed no fluorescence signal on the live MMMT539 cancer cell line by fluorescence or confocal microscopy.
  • Anti-SAS1 B C-terminal monoclonal antibodies 6C1 and 7H2 showed no fluorescence signal on the surface of live uterine tumor cells like the anti- CABYR monoclonal antibody 3A4, suggesting a lack of SAS1 B C-terminal epitope between residues 280 to 431 being exposed to the cell surface.
  • Live MPanc96 cells pancreatic ductal adenocarcinoma cell line
  • different anti-SAS1 B monoclonal antibodies as well as DAPI stain for nucleus localization.
  • Cells were fixed and permeabilized after primary and secondary antibody incubation thus fluorescence staining observed represents surface localized SAS1 B.
  • Monoclonal antibodies 1 H3/1 K2 and 2H2/2K5 were used, as well as normal rabbit or mouse IgG's as a negative control. All primary antibodies used at a concentration of 20 ug/mL.
  • SAS1 B monoclonal antibodies directed to the N-terminus of SAS1 B and monoclonal antibodies directed to the C-terminus of SAS1 B show differential cell surface staining patterns in MPanc96 cells. Monoclonal antibodies that recognize the N-terminus of SAS1 B showed punctate SAS1 B surface expression while monoclonal antibodies directed against the C-terminus of SAS1 B showed homogenous SAS1 B expression.
  • One hypothesis is that both N- and C- termini are exposed extracellularly where the N-terminus has a short peptide exposed compared to a more bulky C-terminus of the protein which causes staining to appear more homogenous.
  • SAS1 B-CT rabbit monoclonal antibodies were used in live immunofluorescent staining of SAS1 B in SNU 539 cancer cell lines. Acutase split cells were plated on plain coverslips at 7000 cells/well. The cells were blocked with 5% NGS media and chilled in 0.1 % azide-containing media. The cells were incubated with undiluted rabbit monoclonal antibody supernatants and Rb2 IM serum at 1 :50 dilution for 2 hours on ice and then washed with azide buffer. The secondary antibody (goat anti-rabbit Alexa 488, 1 :500 dilution) was added in blocking buffer and incubated 1 hour on ice in the dark.
  • the cells were washed with azide buffer and fixed with 4% PFA for 15 mins at room temperature.
  • the cells were mounted in antifade reagent with DAPI.
  • DAPI antifade reagent
  • HEK293T cells were transfected with human SAS1 B SV-A full length cDNA for 66 h.
  • Immunoprecipitation ("IP") of SAS1 B was completed with 7H2 monoclonal antibodies. Detection of the SAS1 B immunoprecipitation product was also completed with HRP peroxidase conjugated mouse anti-His mAb at 1 :3000 or 1 :5000 dilution.
  • HEK293T cells were seeded at 250,000. The cells were transfected with 20 mg of SAS1 B-SVA plasm id into each of the two T-75 flasks (at -90% confluency) for -66 h at 37°C and 5% C0 2 .
  • the magnetic bead/antibody complex was washed with PBST once, followed by incubation with 300 ml_ antigen extract for 20 min. The post immunoprecipitation sample was saved for comparison. Three 200 ml_ washes with washing buffer+protease inhibitor. The fourth wash used only 100 ml_ of wash buffer+protease inhibitor.
  • the magnetic beads were transferred to a new micro-centrifuge tube. 1X Lamelli buffer was added to bead/antibody/antigen complex for subsequent analysis. The sample was boiled at 98°C for 10 min in 1X Lamelli buffer and the supernatant transferred to a new tube. The immunoprecipitation sample was analyzed by western blot and Coomassie blue staining.
  • the SDS-PAGE samples (pre-IP, IP, and post IP extracts) were resolved with 12% TGX Criterion gels at 30 milliamps for 1 hr. Coomassie stain was added to determine the profile of IP samples. Following SDS-PAGE resolution, bands were transferred to nitrocellulose membrane at 1 Amp for 1 h at 5°C. The membrane was blocked with 5% dry milk/PBST, and incubated with mouse anti-His monoclonal antibody conjugated with HRP for 1 hr at RT at 1 :3000 or 1 :5000 dilution. The membranes were imaged with TMB solution.
  • the western blot showed the SAS1 B antigen was pulled down by monoclonal antibody 7H2.
  • the 3A7 mAb and protein G bead did not pull down rSASI B.
  • the remaining SAS1 B in the post IP (“PIP") sample was compared with starting material ("SM”).
  • the negative control mAb 3A7 did not pull down the antigen.
  • 3A7 showed no reduction of SAS1 B in post IP samples.
  • Parental line clone hybridoma cell lines SAS1 B 6C1 , SAS1 B 7H2 were sequenced.
  • RT-PCR was carried out using 5' RACE and gene specific reverse primers which amplify mouse immunoglobulin heavy chain (lgG1 ) and light chain (kappa) variable region sequences.
  • the specific bands were excised and cloned into pCR-Blunt ll-TOPO® vector for sequencing, and the constructs were transformed into E. coli. 16-24 colonies of each chain were picked and PCR screened for the presence of amplified regions prior to sequencing; additional colonies were picked as necessary (see Table 5).
  • PCR positive clones were selected (8 heavy chain and 8 kappa chain) and sequenced. DNA sequences were analyzed by BLAST and SnapGene to confirm homology to mouse antibody sequences.
  • the DNA sequences we re determined for eight each of SAS1 B 6C1 and 7H2 lgG1 heavy chain, five SAS1 B 6C1 kappa chain and seven SAS1 B 7H2 kappa chain variable regions.
  • a fourth sequence (K4) contains a single "T” to "A" mutation at position 329, which would change a phenylalanine to a tyrosine in the amino acid sequence.
  • the remaining 3 sequences (K9, K1 & K6) contain a four amino acid deletion which would shift the reading frame, so that the "RADA” sequence which identifies the beginning of the kappa constant region is not translated.
  • the consensus DNA sequence for SAS1 B 7H2 kappa chain variable region is shown below (the bolded "T" at position 96 can optionally be a "C”; The bolded T at position 329 can optionally be an "A”):
  • Antibody 2H2/2K5 was sequenced. The consensus DNA sequence of 2H2 rabbit IgG variable heavy chain is shown below.
  • SEQ ID NO : 79 The translation of consensus DNA sequence for 1 H2 rabbit IgG heavy constant chain is shown below.
  • RACE Rapid Amplification of cDNA Ends
  • amino acid sequence of the heavy chain variable region of 3F2 is as follows:
  • the nucleotide sequence of the heavy chain variable region of 3F2 is as follows:
  • CDR1 of the 3F2 heavy chain variable region is GFTFINYW (SEQ ID NO:20).
  • CDR2 of the 3F2 heavy chain variable region is lYPGKSDI (SEQ ID NO:21 ).
  • CDR 3 of the 3F2 heavy chain variable region is TRGGAMDY (SEQ ID NO:22).
  • amino acid sequence of the light chain variable region of 3F2 is as follows:
  • the nucleotide sequence of the light chain variable region of 3F2 is as follows:
  • CDR1 of the light chain variable region of 3F2 is QSLLYSSDQKNY (SEQ ID NO:23).
  • CDR2 of the light chain variable region is FAS.
  • CDR3 of the light chain variable region is QQHYNTPLT(SEQ ID NO:24).
  • amino acid sequence of the 6B1 heavy chain variable region is shown below:
  • the nucleotide sequence of the 6B1 heavy chain variable region is shown below:
  • CDR1 of the 6B1 heavy chain variable region is GFTFINYW (SEQ ID NO:20).
  • the CDR2 of the 6B1 heavy chain variable region is lYPGKSDI (SEQ ID NO:21 ).
  • the CDR3 of the 6B1 heavy chain region is TRGGAMDY (SEQ ID NO:22).
  • amino acid sequence of the 6B1 light chain variable region is shown below:
  • the nucleotide sequence of the 6B1 light chain variable region is shown below:
  • CDR1 of 6B1 light chain variable region is QSLLYSSDQKNY (SEQ ID NO:23).
  • the CDR2 of the 6B1 light chain variable region is FAS.
  • the CDR3 of the 6B1 light chain variable region is QQHYNTPLT (SEQ ID NO:24).
  • Recombinant rabbit clones 3H4/3K5, 5H2/5K1 , 6H2/6K1 , 7H1 /7K3, 9H1/9K5 and 31 H1/31 K1 were sequenced and analyzed. Variable region alignments of both DNA sequences and translated DNA sequences show that all six clones are different in sequence. It is likely that the six recombinant rabbit clones recognize SAS1 B via different epitopes.
  • the nucleotide sequences of the variable region of the six heavy chains were aligned, from the putative ATG start codon to the beginning of the consensus constant region.
  • the translated variable sequences of the heavy chains were also aligned, from the initial methionine amino acid to the beginning of the constant region.
  • variable region of 3H4 rabbit IgG heavy chain is shown below.
  • variable region of the 3H4 rabbit IgG heavy chain was translated into the amino acid sequence shown below.
  • variable region of 5H2 rabbit IgG heavy chain was translated into the amino acid sequence shown below:
  • variable region of 6H2 rabbit IgG heavy chain i s s h own b e l ow .
  • variable region of 7H1 rabbit IgG heavy chain is shown below:
  • variable region of 31 H1 rabbit IgG heavy is shown below:
  • nucleotide sequences of the variable region of the six kappa chains were aligned, from the putative ATG start codon to the beginning of the consensus constant region.
  • the translated variable sequences of the heavy chains were also aligned, from the initial methionine amino acid to the beginning of the constant region.
  • variable region of 3K5 rabbit kappa chain is shown below. 1 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCC 61 ACATTTGCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACA
  • variable region of 5K1 rabbit kappa chain The consensus sequence of variable region of 5K1 rabbit kappa chain shown below:
  • variable region of 6K1 rabbit kappa chain The consensus sequence of variable region of 6K1 rabbit kappa chain shown below:
  • variable region of 7K3 rabbit kappa chain shown below. 1 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCC
  • variable region of 9K5 rabbit kappa chain is shown below: 1 ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCC
  • variable region of 31 K1 rabbit kappa chain is shown below:
  • the cells were blocked with 5% NGS in chilled media + 0.1 % sodium azide (blocking 2 solution) for 30 min on ice.
  • the primary antibody (1 H3/1 K2, 2H2/2K5 or control) was added at 20ug/ml_ in blocking 2 solution for 2 hours on ice at 4°C.
  • the cells were washed 3x for 5 minutes each on ice.
  • the secondary antibody (goat anti-Ms/Rb Alexa488 (1 :500)) was added in blocking 2 solution for 1 hour in dark on ice at 4°C.
  • the cells were washed 3x for 5 min each on ice.
  • the cells were fixed with 4% PFA-PBS for 15 min in the dark at RT.
  • the cells were washed 2x for 5 min each at RT.
  • the cells were stained with DAPI stain (1 : 1000) for 10 min at RT. The cells were washed 2x for 5 min each at RT. The cells were treated with PROLONG® gold antifade. 2H2/2K5 showed about 60-70% cell staining for MPanc96 cells, but only about 5% cell staining for 366 cells. About 10- 25% of the 1 H3/1 K2 treated MPanc96 and 366 cells stained for SASB1 .
  • the cell surface density of SAS1 B was examined in SNU cells (endometrial tumor cells), 3T3(T) cells (positive control cells transfected with SAS1 B), and K562 cells (myelogenous leukemia) with antibody 2H2/2K5 (also called RCT-7 antibody). The results are shown in Figure 2.
  • SNU cells endometrial tumor cells
  • 3T3(T) cells positive control cells transfected with SAS1 B
  • K562 cells myelogenous leukemia with antibody 2H2/2K5 (also called RCT-7 antibody).
  • the SNU cells have about 8,000 copies of SAS1 B on their surface while the 3T3(T) cells and K562 cells have significantly fewer copies of SAS1 B.
  • the results are shown in Figure 3.
  • the RCT-7 antibody had similar results to the control antibody.
  • FACS analysis was performed using several different cell lines, antibody RCT- 7 and a control antibody (Novus propeptide antibody).
  • the cell lines were 3T3(T) (a positive control), M 1X3 cells (tumor cells), H522 cells (tumor cells), H23 cells (tumor cells), SNU-539 cells (tumor cells), HRE cells (normal cells) and HUVEC cells (normal cells).
  • Antibody RCT-7 results were similar to and tracked the control antibody.

Abstract

La présente invention concerne des anticorps anti-SAS1B, des fragments de liaison à l'antigène de ceux-ci, des conjugués anticorps-médicament et leurs méthodes d'utilisation.
PCT/US2017/019052 2016-02-23 2017-02-23 Anticorps anti-sas1b et ses méthodes d'utilisation WO2017147247A1 (fr)

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