# Patent

## Title
Anti-EphA2 antibodies and methods of use thereof

## Abstract

Antibodies that bind to tumor associated antigen CD44 or to tumor associated antigen EphA2, are disclosed herein, as well as related compositions and methods of use. Methods of use encompass cancer therapies, diagnostics, and screening methods.

## Background
CROSS-REFERENCE
This application is a continuation of U.S. application Ser. No. 13/810,638, filed Apr. 30, 2013, which claims the benefit of International Application Serial No. PCT/US2011/045069, filed Jul. 22, 2011, which claims the benefit of U.S. Provisional Patent Application No. 61/366,823, filed Jul. 22, 2010, each of which application is incorporated herein by reference in its entirety.


STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with government support under grant no. CA058207 awarded by the National Institutes of Health. The government has certain rights in the invention.


INTRODUCTION
Extensive studies of cancer transcriptional patterns have led to the discovery of molecular targets to distinguish the malignant from the benign and the most aggressive cancers from those that are less aggressive. Cancers often overexpress a number of proteins, including certain cell surface antigens, e.g., cell surface receptors. Antibodies that bind such overexpressed cell surface antigens can facilitate detection and treatment of such cancers. A number of approaches have been utilized to generate antibodies to cancer cell surface receptors which can be used as potential therapeutics. Identification of overexpressed cell surface receptors and antibodies which bind them provide a route to the development of cancer therapies, especially for those cancer subtypes with poor prognosis and resistance to traditional therapies. CD44 and EphA2 are two such overexpressed cell surface receptors, and are known from transcriptional profiling and proteomic analysis to be overexpressed in basal breast cancers.
Antibodies specific to cell surface receptors overexpressed on a number of cancers have been utilized for development of targeted immunotherapeutics. For example, HER2, CD20, and EGFR are overexpressed on a number of tumors and antibodies recognizing these receptors have been developed to treat metastatic breast cancer (trastuzamab), lymphoma (rituximab), and colorectal cancer (cetuximab). Several therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor, but that also undergo internalization into the cell upon binding.
SUMMARY
Antibodies that bind to tumor associated antigen CD44 or to tumor associated antigen EphA2, are disclosed herein, as well as related compositions and methods of use. Methods of use encompass cancer therapies, diagnostics, and screening methods. In certain embodiments, antibodies bind mammalian cell surface antigen (e.g., yeast displayed mammalian cell surface antigen), in others they are endocytosed upon binding to mammalian cells.
In one embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of CD44 that is specifically bound by antibody F2-1A6 or that competes with antibody F2-1A6 for binding to CD44. Said antibody may, when bound to CD44 on the surface of a living mammalian cell, be endocytosed (by the cell). In one embodiment, said antibody comprises a VH CDR1 of F2-1A6, a VH CDR2 of F2-1A6 and a VH CDR3 of F2-1A6. In another embodiment, said antibody comprises a VL CDR1 of F2-1A6, a VL CDR2 of F2-1A6, and a VL CDR3 of F2-1A6. In yet another embodiment, said antibody competes for binding to an epitope of CD44 with an antibody comprising, a full length VH of F2-1A6, and a full length VL of F2-1A6.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of CD44 that is specifically bound by antibody F2-1H9 or that competes with antibody F2-1H9 for binding to CD44. Said antibody may, when bound to CD44 on the surface of a living mammalian cell, be endocytosed. In one embodiment, said antibody comprises a VH CDR1 of F2-1H9, a VH CDR2 of F2-1H9, and a VH CDR3 of F2-1H9. In another embodiment, said antibody comprises a VL CDR1 of F2-1H9, a VL CDR2 of F2-1H9, and a VL CDR3 of F2-1H9. In yet another embodiment, said antibody comprises a full length VH of F2-1H9 and a full length VL of F2-1H9.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of CD44 that is specifically bound by an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3; or that competes with an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3 for binding to CD44. In one embodiment, said antibody comprises: a VH CDR1 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; a VH CDR2 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; and a VH CDR3 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. In another embodiment said antibody comprises: a VL CDR1 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; a VL CDR2 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; and a VL CDR3 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. In another embodiment said antibody comprises: a full length VH of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3; and a full length VL of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of EphA2 that is specifically bound by antibody 2D6 or that competes with antibody 2D6 for binding to EphA2. Said antibody may, when bound to EphA2 on the surface of a living mammalian cell, be endocytosed.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of EphA2 that is specifically bound by antibody D2-1A7 or that competes with antibody D2-1A7 for binding to EphA2. Said antibody may, when bound to EphA2 on the surface of a living mammalian cell, be endocytosed.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of EphA2 that is specifically bound by antibody D2-1A9 or that competes with antibody D2-1A9 for binding to EphA2. Said antibody may, when bound to EphA2 on the surface of a living mammalian cell, be endocytosed.
In another aspect, an isolated monoclonal antibody is provided that specifically binds an epitope of EphA2 that is specifically bound by antibody D2-1B1 or that competes with antibody D2-1B1 for binding to EphA2. Said antibody may, when bound to EphA2 on the surface of a living mammalian cell, be endocytosed.
In another embodiment, an isolated monoclonal antibody is provided that specifically binds an epitope of EphA2 that is specifically bound by an antibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11 or that competes with an antibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11 for binding to EphA2. In one embodiment, said antibody comprises: a VH CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; a VH CDR2 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; and a VH CDR3 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. In another embodiment, said antibody comprises: a VL CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; a VL CDR2 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; and a VL CDR3 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
In certain embodiments, any of the aforementioned monoclonal antibodies is a single chain Fv (scFv), IgG, Fab, (Fab′)2, or (scFv′)2. Any of said antibodies may be labeled and/or be conjugated to an anti-cancer agent.
In one embodiment, a lipidic nanoparticle comprising a surface and an interior space is provided, said interior space comprising an anti-cancer agent, wherein any one or more of the aforementioned isolated antibodies is attached to the surface of said lipidic nanoparticle. In one embodiment, when the lipidic nanoparticle is contacted with a cell expressing cell surface EphA2 or cell surface CD44, said antibody binds to the cell surface EphA2 or cell surface CD44 and the lipidic nanoparticle is endocytosed.
In one embodiment, a composition is provided comprising a pharmaceutically acceptable carrier and any one of the aforementioned antibodies or any one of the aforementioned lipidic nanoparticles. Said composition may be formulated for parenteral administration. Alternatively, said composition may be formulated for intravenous, intrathecal, or intraventricular administration or for convection enhanced delivery. In one embodiment, a kit comprising the composition is provided.
In another embodiment, a method of treating a subject having cancer is provided, said method comprising administering to said subject an amount of any one of the aforementioned antibodies or any one of the aforementioned lipidic nanoparticles, wherein said amount is sufficient to slow the growth of the cancer. In one embodiment said antibody is internalized into a cancer cell
In one embodiment, a method of detecting a cancer cell in a subject is provided, comprising contacting an antibody of any one of the aforementioned antibodies with a cell of said subject suspected of being cancerous and detecting said antibody bound to said cell.
In another embodiment, an isolated nucleic acid is provided, comprising a nucleotide sequence encoding an amino acid sequence of: a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone F2-1A6; a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone F2-1A6; a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone F2-1H9; a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone F2-1H9; a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3; or a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3. In one embodiment, said nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of: a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone F2-1A6 and a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone F2-1A6; a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone F2-1H9 and a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone F2-1H9; or a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3 and a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody selected from E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3.
In another embodiment, an isolated nucleic acid is provided, comprising a nucleotide sequence encoding an amino acid sequence of: a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone 2D6, a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone 2D6, a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone D2-1A7, a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone D2-1A7, a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone D2-1A9, a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone D2-1A9, a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody from clone D2-1B1, a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody from clone D2-1B1; a VH comprising a VH CDR1, a VH CDR2 and a VH CDR3 of an antibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11; a VL comprising a VL CDR1, a VL CDR2 and a VL CDR3 of an antibody selected from A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11; or an isolated nucleic acid comprising nucleotide sequences encoding the amino acid sequences of 1) and 2); 3) and 4); 5) and 6); 7) and 8); or 9) and 10).
In one embodiment, an expression vector is provided that comprises any of the aforementioned nucleic acids. In another embodiment, a recombinant host cell (a “genetically modified host cell”) is provided comprising said vector. In one embodiment the cell expresses an anti-EphA2 antibody or an anti-CD44 antibody.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Display of antigen domains on the surface of yeast. Panel A, The extracellular domain (ECD) of receptor EphA2 was displayed on the yeast surface and was recognized by an anti-EphA2 antibody and recombinant mouse Ephrin A1 (R&D, Minneapolis) as determined by flow cytometry analysis. Panel B, The link domain of CD44 (domain 1, or D1) was displayed on the yeast surface and recognized by an anti-CD44 rabbit monoclonal antibody as determined by flow cytometry analysis. Both anti-EphA2 and anti-CD44 antibodies did not recognize an irrelevant protein displayed on the yeast surface.
FIG. 2. Recovery of phage antibodies from yeast displayed antigens. Panel A, Comparison of the recovery of a phage antibody known to bind EphA2 (2D6) from yeast displaying EphA2 ECD (Y-EphA2) versus yeast displaying an irrelevant protein (Y-CON). A total of 1011 anti-EphA2 phage antibody 2D6 were incubated with each of the yeast displayed antigens. Panel B, Impact of the elution buffer on the titer of EphA2 phage antibody eluted from the surface of yeast displaying EphA2 ECD. A total of 1011 anti-EphA2 phage antibody 2D6 were incubated with each of the yeast displayed antigen proteins prior to elution. Panel C, The impact of input phage titer on the eluted phage titer. The indicated titer of anti-EphA2 phage antibody 2D6 was incubated with yeast displayed EphA2 ECD or an irrelevant yeast displayed protein and the titer of eluted phage determined.
FIG. 3. Strategy for selecting internalizing antigen specific phage antibodies. Panel A, After two rounds of selection for internalization on the basal breast cancer cell line MDAMB231, the pool of phage antibodies was first incubated with irrelevant control yeast to remove any yeast binding antibodies followed by panning on yeast displaying either EphA2 ECD or CD44 domain 1. Panel B, The binding signal of the polyclonal phage antibody pool to EphA2 ECD or CD44 domain 1 after 2 rounds of panning was measured by using flow cytometry. The irrelevant control yeast was stained with unselected phage antibody library (R0), round 1 (R1) and round 2 (R2) polyclonal phage. Panel C, Frequency of antigen specific phage antibodies after one and two rounds of selection on yeast displayed antigen. Binding frequency was determined by analyzing 96 randomly picked phage antibodies for binding to yeast displayed antigen by flow cytometry. The induced yeast cells displaying an irrelevant protein, EphA2-ECD or CD44 domain 1 were stained with un-selected phage library (R0) and polyclonal phages from R1 and R2 respectively. The un-induced yeast cells (yeast only), irrelevant phage antibody (phage control) and the un-selected phage antibody library were used as control.
FIG. 4. Binding specificity of monoclonal phage antibodies from yeast antigen biopanning. Panel A, Binding of monoclonal phage antibodies to yeast displayed antigen domains as determined by flow cytometry. The induced yeast cells displaying an irrelevant protein (Y-CON), EphA2-ECD (Y-EphA2 ECD), CD44 link domain (Y-CD44 ECD D1) and CD44 full length ECD (Y-CD44 ECD) were stained with monoclonal phage antibodies isolated from Y-EphA2 and Y-CD44 D1 selections. Panel B shows the binding of monoclonal phage antibodies to MDAMB231 cells as determined by flow cytometry. Panel C shows the differential binding of monoclonal phage antibodies to breast cancer cell lines including luminal breast cancer cell lines SUM52PE and MCF7, and basal breast cancer cell line MDAMB231.
FIG. 5. Characterization of scFv antibodies by western-blot and flow cytometry. Panel A, Anti-EphA2 scFv 2D6 or D2-1A7 and anti-CD44 scFv F2-1A6 were used to immunoprecipitate their target antigen from MDAMB231 cells. Antigen was detected by Western blotting using either anti-EphA2 antibody D7 (Upstate Biotech, Billerica) or anti-CD44 antibody Ab-4 (NeoMarkers, Fremont). Panel B, EphA2 antibodies D2-1A7, D2-1A9, and 2D6 compete with ephrin A1 for binding to MDAMB231 cells. The ability of phage antibodies D2-1A7, D2-1A9, and 2D6 that bind to MDAMB231 cells in the presence of increasing concentrations of the EphA2 ligand ephrin A1 was determined by flow cytometry.
FIG. 6. Phage antibodies specific to EphA2 and CD44 are endocytosed by MDAMB231 cells. Cultured cells were incubated with irrelevant phage (panel A), anti-EphA2 phage D2-1A7 (panel B) and D2-1A9 (panel C), anti-CD44 phage F2-1A6 (panels D and E) for 3 hr at 37° C. followed by Glycine buffer wash. Endocytosis was determined by detection of intracellular phage with anti-fd antibody, and analyzing by confocal microscopy.
FIG. 7. Amino acid sequences of full length VH and VL of antibodies specific for CD44 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refers to the full length variable region.
FIG. 8. Amino acid sequences of full length VH and VL of antibodies specific for EphA2 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refers to the full length variable region.
FIGS. 9A and 9B. Amino acid sequences and encoding nucleic acid sequences for full length antibodies from clone 2D6.
FIGS. 10A and 10B. Amino acid sequences and encoding nucleic acid sequences for full length scFv antibodies from clone D2-1A7.
FIGS. 11A and 11B. Amino acid sequences and encoding nucleic acid sequences for full length scFv antibodies from clone D2-1A9.
FIGS. 12A and 12B. Amino acid sequences and encoding nucleic acid sequences for full length scFv antibodies from clone D2-1B1.
FIGS. 13A and 13B. Amino acid sequences and encoding nucleic acid sequences for full length scFv antibodies from clone F2-1A6.
FIGS. 14A and 14B. Amino acid sequences and encoding nucleic acid sequences for full length scFv antibodies from clone F2-1H9.
FIGS. 15A and 15B. Amino acid (SEQ ID NO:90) and encoding nucleic acid (SEQ ID NO:91) sequences for full length scFv antibodies from clone E8H11.
FIGS. 16A and 16B. Amino acid (SEQ ID NO:58) and encoding nucleic acid (SEQ ID NO:59) sequences for full length scFv antibodies from clone E8H7.
FIGS. 17A and 17B. Amino acid (SEQ ID NO:92) and encoding nucleic acid (SEQ ID NO:93) sequences for full length scFv antibodies from clone E8G12.
FIGS. 18A and 18B. Amino acid (SEQ ID NO:63) and encoding nucleic acid (SEQ ID NO:64) sequences for full length scFv antibodies from clone E8F11.
FIGS. 19A and 19B. Amino acid (SEQ ID NO:72) and encoding nucleic acid (SEQ ID NO:73) sequences for full length scFv antibodies from clone E8C9.
FIGS. 20A and 20B. Amino acid (SEQ ID NO:85) and encoding nucleic acid (SEQ ID NO:86) sequences for full length scFv antibodies from clone D6G9.
FIGS. 21A and 21B. Amino acid (SEQ ID NO:127) and encoding nucleic acid (SEQ ID NO:128) sequences for full length scFv antibodies from clone D6D3.
FIGS. 22A and 22B. Amino acid (SEQ ID NO:129) and encoding nucleic acid (SEQ ID NO:130) sequences for full length scFv antibodies from clone A3H9.
FIGS. 23A and 23B. Amino acid (SEQ ID NO:135) and encoding nucleic acid (SEQ ID NO:136) sequences for full length scFv antibodies from clone A3G3.
FIGS. 24A and 24B. Amino acid (SEQ ID NO:137) and encoding nucleic acid (SEQ ID NO:138) sequences for full length scFv antibodies from clone A3D10.
FIGS. 25A and 25B. Amino acid (SEQ ID NO:305) and encoding nucleic acid (SEQ ID NO:306) sequences for full length scFv antibodies from clone A3D1.
FIGS. 26A and 26B. Amino acid (SEQ ID NO:307) and encoding nucleic acid (SEQ ID NO:308) sequences for full length scFv antibodies from clone A3C8.
FIGS. 27A and 27B. Amino acid (SEQ ID NO:309) and encoding nucleic acid (top strand—SEQ ID NO:310; bottom strand—SEQ ID NO:416) sequences for full length scFv antibodies from clone 1A3.
FIGS. 28A and 28B. Amino acid (SEQ ID NO:311) and encoding nucleic acid (top strand—SEQ ID NO:312; bottom strand—SEQ ID NO:417) sequences for full length scFv antibodies from clone 1A5.
FIGS. 29A and 29B. Amino acid (SEQ ID NO:313) and encoding nucleic acid (top strand—SEQ ID NO:314; bottom strand—SEQ ID NO:418) sequences for full length scFv antibodies from clone 1A8.
FIGS. 30A and 30B. Amino acid (SEQ ID NO:315) and encoding nucleic acid (top strand—SEQ ID NO:316; bottom strand—SEQ ID NO:419) sequences for full length scFv antibodies from clone 1A12.
FIGS. 31A and 31B. Amino acid (SEQ ID NO:317) and encoding nucleic acid (top strand—SEQ ID NO:318; bottom strand—SEQ ID NO:420) sequences for full length scFv antibodies from clone 1B2.
FIGS. 32A and 32B. Amino acid (SEQ ID NO:319) and encoding nucleic acid (top strand—SEQ ID NO:320; bottom strand—SEQ ID NO:421) sequences for full length scFv antibodies from clone 1C2.
FIGS. 33A and 33B. Amino acid (SEQ ID NO:321) and encoding nucleic acid (top strand—SEQ ID NO:322; bottom strand—SEQ ID NO:422) sequences for full length scFv antibodies from clone 1C7.
FIGS. 34A and 34B. Amino acid (SEQ ID NO:323) and encoding nucleic acid (top strand—SEQ ID NO:324; bottom strand—SEQ ID NO:423) sequences for full length scFv antibodies from clone 1D8.
FIGS. 35A and 35B. Amino acid (SEQ ID NO:325) and encoding nucleic acid (top strand—SEQ ID NO:326; bottom strand—SEQ ID NO:424) sequences for full length scFv antibodies from clone 15H11.
FIGS. 36A and 36B. Amino acid (SEQ ID NO:327) and encoding nucleic acid (top strand—SEQ ID NO:328; bottom strand—SEQ ID NO:425) sequences for full length scFv antibodies from clone D1C5.
FIGS. 37A and 37B. Amino acid (SEQ ID NO:329) and encoding nucleic acid (top strand—SEQ ID NO:330; bottom strand—SEQ ID NO:426) sequences for full length scFv antibodies from clone D1D1.
FIGS. 38A and 38B. Amino acid (SEQ ID NO:331) and encoding nucleic acid (top strand—SEQ ID NO:332; bottom strand—SEQ ID NO:427) sequences for full length scFv antibodies from clone H1B8 (also referred to herein as “HB8”).
FIGS. 39A and 39B. Amino acid (SEQ ID NO:333) and encoding nucleic acid (top strand—SEQ ID NO:334; bottom strand—SEQ ID NO:428) sequences for full length scFv antibodies from clone H1C2 (also referred to herein as “HC2”).
FIGS. 40A and 40B. Amino acid (SEQ ID NO:335) and encoding nucleic acid (top strand—SEQ ID NO:336; bottom strand—SEQ ID NO:429) sequences for full length scFv antibodies from clone H1C4 (also referred to herein as “HC4”).
FIGS. 41A and 41B. Amino acid (SEQ ID NO:337) and encoding nucleic acid (top strand—SEQ ID NO:338; bottom strand—SEQ ID NO:430) sequences for full length scFv antibodies from clone H1E3 (also referred to herein as “HE3”).
FIGS. 42A and 42B. Amino acid (SEQ ID NO:339) and encoding nucleic acid (top strand—SEQ ID NO:340; bottom strand—SEQ ID NO:431) sequences for full length scFv antibodies from clone H1F1 (also referred to herein as “HF1”).
FIGS. 43A and 43B. Amino acid (SEQ ID NO:127) and encoding nucleic acid (top strand—SEQ ID NO:128; bottom strand—SEQ ID NO:432) sequences for full length scFv antibodies from clone H1H3 (also referred to herein as “HH3”).
FIG. 44. Amino acid sequences of full length VH and VL of antibodies specific for CD44 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refer to the full length variable region. VH sequences of E8H11 (SEQ ID NO:90); E8H7 (SEQ ID NO:342); E8G12 (SEQ ID NO:343); E8F11 (SEQ ID NO:344); E8C9 (SEQ ID NO:345); D6G9 (SEQ ID NO:346); and D6D3 (SEQ ID NO:347) are provided. VL sequences of E8H11 (SEQ ID NO:348); E8H7 (SEQ ID NO:349); E8G12 (SEQ ID NO:350); E8F11 (SEQ ID NO:351); E8C9 (SEQ ID NO:352); D6G9 (SEQ ID NO:353); and D6D3 (SEQ ID NO:354) are provided.
FIGS. 45A and 45B. Amino acid sequences of full length VH and VL of antibodies specific for CD44 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refer to the full length variable region. VH sequences of D1C5 (SEQ ID NO:355); D1D1 (SEQ ID NO:356); HB8 (SEQ ID NO:357); HC2 (SEQ ID NO:358); HC4 (SEQ ID NO:359); HE3 (SEQ ID NO:360); HF1 (SEQ ID NO:361); and HH3 (SEQ ID NO:347) are provided. VL sequences of D1C5 (SEQ ID NO:362); D1D1 (SEQ ID NO:363); HB8 (SEQ ID NO:364); HC2 (SEQ ID NO:365); HC4 (SEQ ID NO:366); HE3 (SEQ ID NO:367); HF1 (SEQ ID NO:368); and HH3 (SEQ ID NO:354) are provided.
FIG. 46. Amino acid sequences of full length VH and VL of antibodies specific for EphA2 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refer to the full length variable region. VH sequences of A3H9 (SEQ ID NO:369); A3G3 (SEQ ID NO:370); A3D10 (SEQ ID NO:371); A3D1 (SEQ ID NO:372); and A3C8 (SEQ ID NO:373) are provided. VL sequences of A3H9 (SEQ ID NO:374); A3G3 (SEQ ID NO:375); A3D10 (SEQ ID NO:376); A3D1 (SEQ ID NO:377); and A3C8 (SEQ ID NO:378) are provided.
FIG. 47. Amino acid sequences of full length VH and VL of antibodies specific for EphA2 from various clones. The framework and complementary determining regions are also indicated. SEQ ID NOs refer to the full length variable region. VH sequences of 1A3 (SEQ ID NO:379); 1A5 (SEQ ID NO:380); 1A8 (SEQ ID NO:381); 1A12 (SEQ ID NO:382); 1B2 (SEQ ID NO:383); 1C2 (SEQ ID NO:384); 1C7 (SEQ ID NO:385); and 1D8 (SEQ ID NO:386) are provided. VL sequences of 1A3 (SEQ ID NO:387); 1A5 (SEQ ID NO:388); 1A8 (SEQ ID NO:389); 1A12 (SEQ ID NO:390); 1B2 (SEQ ID NO:391); 1C2 (SEQ ID NO:392); 1C7 (SEQ ID NO:393); and 1D8 (SEQ ID NO:394) are provided.



DEFINITIONS
The following abbreviations may be used herein: CDR, complementarity determining region; Fab, antigen binding fragment of immunoglobulin with variable domain and first constant domain; FACS, fluorescent activated cell sorting; IgG, immunoglobulin G; KD, dissociation equilibrium constant; kon, association rate constant; koff, dissociation rate constant; mAb, monoclonal antibody; MFI, mean fluorescent intensity; PBS, phosphate buffered saline; PCR, polymerase chain reaction; scFv, single chain format of antibody variable regions; VH, heavy chain variable region; VL, light chain variable region; TAA, tumor associated antigen; EGFR, epidermal growth factor receptor; ECD, extracellular domain; IMAC, immobilized metal affinity chromatography; ILs, immunoliposomes; IPTG, Isopropyl-β-D-thiogalactopyranoside; 2-MEA, 2-Mercaptoethylamine; DTT, Dithiothreitol; TEA, triethylamine; TBS-T, Tris-buffered saline polysorbate 20/TWEEN® 20; Ni-NTA, Nickel-nitrilotriacetic acid; PE, phycoerythrin; HMEC, human mammary epithelial cell.
As used herein, “EphA2” refers to a member of the receptor tyrosine kinase family that can bind EphrinA ligands, and can also be named “epithelial cell kinase (ECK)”. The term “EphA2” can refer to any naturally occurring isoforms of a EphA2. The amino acid sequence of EphA2 is known and can be found as GenBank Accession No. NP_004422.2.
As used herein, “CD44” refers to receptor for hyaluronic acid (HA), and can also be called “phagocytic glycoprotein I”, “hyaluronate receptor”, or “CD44 antigen”. For example, the term “CD44” can refer to either any one of naturally-occurring isoforms of a CD44. The amino acid sequence of CD44 is known and the longest isoform of CD44 can be found as GenBank Accession No. NP_000601.3.
The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In addition, the amino acids, in addition to the 20 “standard” genetically encodable amino acids, include amino acid analogs. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group. However, the absence of a dash should not be taken to mean that such peptide bonds or covalent bond to a carboxyl or hydroxyl end group is not present, as it is conventional in representation of amino acid sequences to omit such.
“Antibody” encompasses compositions comprising an antigen-binding protein, individually or as a preparation comprising a plurality thereof, having one or more polypeptides that can be genetically encodable by immunoglobulin genes, or fragments of immunoglobulin genes, or that comprise CDRs obtained or derived from immunoglobulins, and which bind an antigen of interest. Light chains are classified as either kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An example of an antibody is one having a structural unit of a tetramer composed of two pairs of polypeptide chains, each pair having one “light” and one “heavy” chain. The N-terminal portion of each chain defines a variable region that mediates antigen binding. The terms variable light chain (VL) and variable heavy chain (VH) refer to light and heavy chains respectively.
“Antibody” also encompasses single-chain antibodies that contain a heavy chain and a light chain linked together as a single polypeptide, each of such linked heavy or light chains nonetheless being referred to herein as a heavy chain or a light chain. Antibody may also refer to heavy chain-only antibodies such as heavy chain antibodies or HCAbs.
As noted above, “antibody” encompasses intact immunoglobulins as well antigen-binding fragments of antibodies. Thus, the term “antibody”, as used herein also includes an antigen-binding portion of an antibody, which can be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Examples include, but are not limited to, Fab′, Fab′2, scFv, nanobodies, unibodies, and diabodies. A “nanobody” refers to the smallest antigen-binding fragment of a single chain antibody, also referred to as a VHH or single-domain antibodies (dAbs).
“Monoclonal antibody” refers to a composition comprising one or more antibodies obtained from a population of substantially homogeneous antibodies, i.e., a population the individual antibodies of which are identical except for any naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site and generally to a single epitope on an antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and does not require that the antibody be produced by any particular method or be the only antibody in the composition.
A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which may be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker (Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883). A number of structures are available for converting the light and heavy polypeptide chains from an antibody V region into a scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. In addition to being diabodies, the scFvs can also be present as tribodies or tetrabodies.
It should be noted that while various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
The term “antibody” encompasses polyclonal and monoclonal antibodies, and further encompasses antibodies of any class (e.g., IgM, IgG, and subclasses thereof). “Antibody” also encompasses hybrid antibodies, bispecific antibodies, heteroantibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which retain antigen binding. “Bispecific antibodies” may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). Heteroantibodies refers to two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity. The antibodies may be conjugated to other moieties, and/or may be bound to a support (e.g., a solid support), such as a polystyrene plate or bead, test strip, and the like.
An immunoglobulin light or heavy chain variable region is composed of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs can be defined based on databases known in the art. See, for example, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987), Lefranc et al. IMGT, the international ImMunoGeneTics information System®. Nucl. Acids Res., 2005, 33:D593-D597 ((worldwideweb(dot)imgt(dot)org)/textes/IMGTScientificChart/), and/or V Base at vbase(dot)mrc-cpe(dot)cam(dot)ac(dot)uk/). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and framework provided by the present disclosure are defined according to Kabat et al., supra, unless otherwise indicated.
An “anti-EphA2 antibody” or “anti-CD44 antibody” refers to an antibody that, specifically binds to EphA2 or CD44, preferably with high affinity. A specific antibody for EphA2 or CD44 does not exhibit comparable binding to other antigens unrelated to EphA2 or CD44 relative to the binding of EphA2 or CD44.
The term “high affinity” when used with respect to an antibody refers to an antibody that specifically binds to (“recognizes”) its target(s) with an affinity (KD) value less than or equal to 10−6 M, less than 10−7 M, less than 10−8 M, preferably less than 10−9 M, less than 10−10 M, or less than 10−11 M. A lower KD value corresponds to a higher binding affinity (i.e., stronger binding) so that a KD value of 10−7 indicates a higher binding affinity than a KD value of 10−6.
An “antigen-binding site” or “binding portion” refers to a part of an antibody molecule (e.g. fragment of an immunoglobulin molecule or scFv) that participates in immunoreactive antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and/or light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions” or “FRs”. Thus, the term “FR” refers to amino acid sequences that are naturally found between and adjacent to hypervariable regions in immunoglobulins. In a tertrameric antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding “surface”. This surface mediates recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and/or light chains are referred to as “complementarity determining regions” or “CDRs” and are determined, for example based on Kabat et al., supra.
A “F2-1A6 antibody” refers to an antibody expressed by clone F2-1A6 or to an antibody synthesized in other manners, but having the same CDRs and optionally, the same framework regions as the antibody expressed by clone F2-1A6, and having substantially the same antigen binding specificity. Similarly, antibodies 2D6, D2-1A7, D2-1A9, D2-1B1, F2-1H9, and the like (e.g., antibodies E8H11, E8H7, E8G12, E8F11, and E8C9; antibodies D6G9 and D6D3; antibodies D1C5 and D1D1; antibodies HB8, HC2, HC4, HE3, HF1, and HH3; antibodies A3H9, A3G3, A3D10, A3D1, and A3C8; antibodies 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, and 1D8; and antibody 15H11) refer to antibodies expressed by the corresponding clone(s) and/or to antibodies synthesized in other manners, but having the same CDRs and optionally, the same framework regions as the referenced antibodies, and having substantially the same antigen binding specificity. The CDRs of these antibodies are defined by Kabat et al., supra, as shown in FIGS. 7, 8, and 44-47, and tables below.
An “epitope” is a site on an antigen (e.g. a site on the EphA2 extracellular domain (ECD)) to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by folding (e.g., tertiary folding) of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a linear or spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). Several commercial laboratories offer epitope mapping services. Epitopes bound by an antibody immunoreactive with a membrane associated antigen can reside on the surface of the cell (e.g. in the extracellular region of a transmembrane protein), so that such epitopes are considered cell-surface accessible, solvent accessible, and/or cell-surface exposed.
“Isolated” refers to an entity of interest (e.g., a protein, e.g., an antibody) that is in an environment different from that in which the entity may naturally occur. An “isolated” entity is separated from all or some of the components that accompany it in nature and may be substantially enriched. “Isolated” also refers to the state of an entity separated from all or some of the components that accompany it during manufacture (e.g., chemical synthesis, recombinant expression, culture medium, and the like). A subject antibody can be substantially pure. “Substantially pure” can refer to compositions in which at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99%, of the total composition is the entity of interest (e.g., a subject antibody).
The phrases “specifically bind(s) to,” “specific for,” “immunoreactive” and “immunoreactivity,” and “antigen binding specificity,” when referring to an antibody, refer to a binding reaction with an antigen which is highly preferential to the antigen or a fragment thereof, so as to be determinative of the presence of the antigen in the presence of a heterogeneous population of antigens (e.g., proteins and other biologics, e.g., in a tissue). Thus, under designated immunoassay conditions, the specified antibodies bind to a particular antigen and do not bind in a significant amount to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, anti-CD44 antibodies can specifically bind to CD44, and do not exhibit comparable binding (e.g., do not exhibit detectable binding) to other proteins present in a tissue sample. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
DETAILED DESCRIPTION
Antibodies that specifically bind to tumor associated antigen CD44 and antibodies that specifically bind to tumor associated antigen EphA2 are disclosed herein, as well as related compositions and methods of use thereof. Methods of use encompass cancer therapies, diagnostics, and screening methods.
Where the antibodies are specific for CD44, the antibodies contain at least one, two, or all three CDRs of the VH of the antibody from (e.g., obtained by the expression of) clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. The antibodies also encompass those containing at least one, two, or all three CDRs of the VL of the antibody from clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. The antibodies also encompass those containing at least one, two, or all three CDRs independently selected from each of the VL and the VH of an antibody from clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. Alternately the antibodies compete for binding to CD44 with (e.g., bind to the same epitope as) and antibody from clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.
Where the antibodies are specific for EphA2, the antibodies contain at least one, two or all three heavy chain (VH) complementarity determining region(s) (CDR(s)) of an antibody from clone 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies also encompass those containing at least one, two or all three light chain (VL) complementarity determining region(s) (CDR(s)) of an antibody from clone 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies also encompass those containing at least one, two, or all three CDRs independently selected from each of the VL and the VH of an antibody from clone 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. Alternately the antibodies compete for binding to EphA2 with (e.g., bind to the same epitope as) and antibody from clone 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
An antibody of the present disclosure may also contain all VH CDRs and/or VL CDRs of an antibody from clone F2-1A6, F2-1H9, 2D6, D2-1A7, D2-1A9, D2-1B1, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies may contain full-length VH chains of an antibody from clone F2-1A6, F2-1H9, 2D6, D2-1A7, D2-1A9, D2-1B1, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The antibodies can also or alternatively contain full-length VL chains of an antibody from clone F2-1A6, F2-1H9, 2D6, D2-1A7, D2-1A9, D2-1B1, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
The antibody may be a full length antibody (i.e., an antibody comprising at least one full-length VH sequence and at least one full length VL sequence) or a fragment such as a single chain Fv (scFv), a Fab, a (Fab′)2, an (ScFv)2, and the like. The antibody may be an IgG (e.g., IgG2) or any other isotype, or may be a bispecific antibody.
The antibodies may be conjugated, such as to an anti-cancer drug, a label, or to a moiety that improves or promotes serum half-life (e.g. poly(ethylene glycol) (PEG)), endocytosis, or another biological function or characteristic. The antibody may also be in a composition comprising a pharmaceutically acceptable excipient, e.g., a composition suitable for injection (e.g., in a unit dosage formulation). The present disclosure also provides compositions that include one or more different antibodies selected from the antibodies described herein and/or antibodies comprising one or more CDRs from these antibodies, and/or one or more antibodies comprising mutants or derivatives of these antibodies. The composition may include one or more antibodies, such as F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
Methods of the present disclosure include those that provide for administering one or more subject antibodies as disclosed herein in an amount effective to treat a subject having cancer expressing the antigen(s) specifically bound by the subject antibody or antibodies. The antibodies provided by this disclosure can also be used for diagnosis/prognosis or imaging of cancer.
Nucleic acids provided herein encode one or more antibodies that are described herein. Host cells containing such nucleic acids are also provided herein, as well as those that produce the subject antibodies (e.g. by secretion). Kits are also provided for preparing compositions containing the subject antibodies or for carrying out the subject methods.
Antibodies
Preferred antibodies have a high affinity (e.g., exhibit KD values of 10−7 M or lower) to one or more of the tumor associated antigens (TAA), EphA2 or CD44, that are cell-surface exposed on cancer cells during at least some portion of the cell cycle. Antibodies having lower affinity (e.g., having KD values of from 10−5 M to 10−6 M) for EphA2 or CD44 are also contemplated. Cancer cells, for example, include those derived from breast cancer cells (e.g. MDAMB231) and others. The subject antibodies include those that are internalized into the cell upon binding to antigen, e.g., an antigen on the surface of a living mammalian cell, e.g. by endocytosis, such as receptor-mediated endocytosis.
The subject antibodies include those that competitively bind to an epitope of CD44 with an antibody from clone F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. The antibodies also encompass those that competitively bind to an epitope on EphA2 with an antibody from clone 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The ability of a particular antibody to recognize the same epitope as another antibody can be determined by the ability of one antibody to competitively inhibit binding of the second antibody (e.g., competitively bind) to the antigen (e.g., as determined by competitive binding assays such as those disclosed in US patent publication No. 20090291085). Competitive inhibition of binding may also be referred to as cross-reactivity of antibodies.
Any of a number of competitive binding assays can be used to measure competition between two antibodies to the same antigen. For example, a sandwich ELISA assay can be used for this purpose. Means of assaying for cross-reactivity are well known to those of skill in the art (see, e.g., Dowbenko et al. (1988) J. Virol. 62: 4703-4711).
An antibody is considered to competitively inhibit binding of a second antibody to an antigen if binding of the second antibody to the antigen is reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%, and often by at least about 90%, in the presence of the first antibody using any of the assays used to assess competitive binding.
This can be ascertained by providing one or more isolated target TAA(s), EphA2 and/or CD44, attached to a solid support and assaying the ability of an antibody to bind to the target TAA or to compete with an antibody described herein for binding to the target TAA (e.g. using surface plasmon resonance).
As noted above, the subject antibodies encompass those that compete with one or more of the following antibodies: 2D6, D2-1A7, D2-1A9, D2-1B1, F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, and 15H11. In addition, the antibodies can have a binding affinity for EphA2 or CD44 comparable to or greater than about 1×10−6 M (i.e., the antibodies can exhibit KD values lower than 10−6 M, e.g., about 10−7 M, 10−8 M, 10−9 M, 10−10 M or an even higher binding affinity such as a KD value of about 10−11 M or 10−12 M).
In a related embodiment, the subject antibodies encompass those that bind to the same epitope as F2-1A6 or F2-1H9 or to the epitope of 2D6, D2-1A7, D2-1A9, or D2-1B1, or to the epitope of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3, or to the epitope of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. Epitope mapping can be performed using pairs of antibodies at concentrations resulting in near saturation and at least 100 relative units (RU) of antibody bound. The amount of antibody bound is determined for each member of a pair, and then the two antibodies are mixed together to give a final concentration equal to the concentration used for measurements of the individual antibodies. Antibodies recognizing different epitopes show an essentially additive increase in the RU bound when injected together, while antibodies recognizing identical epitopes show only a minimal increase in RU. Antibodies may be said to be cross-reactive if, when “injected” together they show an essentially additive increase (e.g. an increase by at least a factor of about 1.4, an increase by at least a factor of about 1.6, or an increase by at least a factor of about 1.8 or 2.)
Epitopes of antibodies can also be ascertained by a number of other standard techniques (see, e.g., Geysen et al (1987) J. Immunol. Meth 102:259-274). This technique involves the synthesis of large numbers of overlapping peptides of EphA2 or CD44. The synthesized peptides are then screened against one or more of the prototypical antibodies (e.g., 2D6, etc.) and the characteristic epitopes specifically bound by these antibodies can be identified by binding specificity and affinity. The epitopes thus identified can be conveniently used for competitive assays as described herein to identify cross-reacting antibodies.
The peptides for epitope mapping can be conveniently prepared using “Multipin” peptide synthesis techniques (see, e.g., Geysen et al (1987) Science 235:1184-1190). Using the known sequence of one or more EphA2 and/or CD44, overlapping polypeptide sequences can be synthesized individually in a sequential manner on plastic pins in an array of one or more 96-well microtest plate(s).
Anti-CD44
Antibodies of the present disclosure include those that specifically bind CD44. Anti-CD44 antibodies encompass those that competitively bind to an epitope (e.g. in domain 1) of CD44 with F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. The epitope bound by anti-CD44 antibodies reside in a contiguous amino acid sequence of CD44 from residue position 21-169, as set forth below.







(SEQ ID NO: 1)


QIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTLPTMAQMEKAL


 


SIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFN


 


ASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQKGEYRTNPEDIY






The residue position numbers of CD44 are determined based on the sequence set forth in GenBank Accession No. NP_000601.3 or UniProt Accession No. P16070.
Antigens that share similar epitopes as CD44 can also be binding targets of subject antibodies. When bound to CD44, a subject antibody can be internalized by the cell expressing the CD44 protein.
Epitopes for which anti-CD44 antibodies have affinity are cell-surface exposed and solvent-accessible on many cancer cells, particularly on the plasma membrane of cells. The epitopes can be accessible to the subject antibodies when the cells are live. For example, the epitopes may be present on cancer cells derived from breast cancers, colon cancers, adenoma, head and neck squamous cell carcinoma (HNSCC), prostate cancers, pancreatic cancers, etc. Cancers cells for which anti-CD44 antibodies have affinity may also be from any cancer that is metastic and/or has metastatic potential.
Additional examples of subject antibodies encompass those that have the same binding specificities and comprise at least two CDRs that each independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of a VH CDR of antibodies shown in FIGS. 7, 44, and 45 (45A and 45B) and in tables below (e.g. VH CDR1 of F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3). The subject antibody can also include all three CDRs from any VH CDRs of each antibody shown in FIGS. 7, 44, and 45, such that each VH CDR in the subject antibody is selected from a single antibody shown in FIGS. 7, 44, and 45, or tables below (e.g., Table 1) and each VH CDR independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of the VH CDR of the antibody shown in FIG. 7, 44, or 45 or tables below (e.g., Table 1). For example, the heavy chain of a subject antibody can contain two VH CDRs or all three VH CDRs of F2-1A6. Alternatively, the heavy chain can contain two VH CDRs or all three VH CDRs of F2-1H9. As further examples, the heavy chain of a subject antibody can contain two VH CDRs or all three VH CDRs of any one of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, and HH3.
Similarly for the light chain, a subject antibody will have the same binding specificity and can contain at least two CDRs that are each independently at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of a VL CDR of each antibody shown in FIGS. 7, 44, and 45 (45A and 45B) or tables below (e.g. VL CDR1 of F2-1A6; e.g., VL CDR1 of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3). The subject antibody can also or alternatively include all three VL CDRs from any of the antibodies shown in FIGS. 7, 44, and 45, or tables below (e.g., Table 2) and each VL CDR independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of the VL CDR of the antibody shown in FIG. 7, 44, or 45, or tables below. For example, the light chain of a subject antibody can contain two VL CDRs or all three VL CDRs of F2-1A6. Alternatively, the light chain can contain two VL CDRs or all three VL CDRs of F2-1H9. As further examples, the light chain of a subject antibody can contain two VL CDRs or all three VL CDRs of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3.
Optionally, antibodies can contain the same (i.e. 100% identity), similar, or different framework sequences (FR) in any of corresponding framework sequences in the heavy or light chain provided in FIGS. 7, 44, and 45 or tables below (e.g., Tables 1 and 2, below), so long as binding specificity is substantially maintained. Where the framework sequences are similar, the framework may be at least about 85%, at least about 86%, at least about 90%, at least about 93%, at least about 96%, at least about 98%, or up to 100% identity to a corresponding framework sequence in any of antibodies shown in FIGS. 7, 44, and 45 or tables below (e.g., Tables 1 and 2, below).
An antibody of the present disclosure may therefore contain a full-length VH and/or full length VL sequence that is at least 80% identity, at least 85%, at least 90%, at least 95%, up to 100% amino acid sequence identity to a full-length VH or VL sequence shown in FIG. 7, 44, or 45 or tables below. For example, a subject antibody can contain the full length VH and/or full length VL of F2-1A6. Alternatively, the subject antibody can contain the full length VH and/or full length VL of F2-1H9. As further examples, a subject antibody can contain the full length VH and/or full length VL of E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3. Examples of antibodies of the present disclosure that bind to CD44 are listed in tables below (e.g., Tables 1 and 2).







TABLE 1





Heavy chain FRs and CDRs of antibodies that specifically bind


CD44


















clone
VH FR1
VH CDR1
VH FR2





F2-1A6
QVQLVQSGAEVKKPGESLKISCKGSGYSFT
SYWIG
WVRQMPGKGLEWMG



(SEQ ID NO: 2)
(SEQ ID
(SEQ ID NO: 4)




NO: 3)



F2-1H9
QVQLQESGGGLVQPGGSLRLSCAASGFTFS
SYRMH
WVRQAPGKGLEWVA



(SEQ ID NO: 5)
(SEQ ID
(SEQ ID NO: 7)




NO: 6)



D1C5
QVQLQQSGGGVVQPGRSLRLSCAASGFTFS
SYGIH
WVRQAPGKGLEWVA



(SEQ ID NO: 28)
(SEQ ID
SEQ ID NO: 7)




NO: 36)



D1D1
QVQLVESGGGLVKPGGSLRLSCAASGFTFS
TYTMS
WARQAPGKGLEWVS



(SEQ ID NO: 24)
(SEQ ID
(SEQ ID NO: 21)




NO: 65)



HB8
QVQLVESGGGVVQPGRSLRLSCAASGFTFS
NYAMI
WVRQAPGKGLEWVS



(SEQ ID NO: 131)
(SEQ ID
(SEQ ID NO: 133)




NO: 132)



HC2
QVQRVESGGGVVQPGRSLRLSCAASGFTFS
SYAMS
WVRQAPGKGLEWVS



(SEQ ID NO: 134)
(SEQ ID
(SEQ ID NO: 133)




NO: 32)



HC4
QVQLVESGGGLVQPGGSLRLSCAASGFTFS
TLAMG
WVRQAPGKGLEWVS



(SEQ ID NO: 74)
(SEQ ID
(SEQ ID NO: 133)




NO: 33)



HE3
QVQLQESGGGLVKPGGSLRLSCEASGFTFS
SYSMN
WVRQAPGKGLEWVS



(SEQ ID NO: 433)
(SEQ ID
(SEQ ID NO: 133)




NO: 37)



HF1
QVQLVESGGGLVQPGGSLRLSCAASGFTFS
NYALI
WVRQAPGKGLEWVS



(SEQ ID NO: 74)
(SEQ ID
(SEQ ID NO: 133)




NO: 78)



HH3
QVQLQESGGGLVQPGGSLRLSCAASGFTFS
SYAMS
WVRQAPGKGLEWVS



(SEQ ID NO: 5)
(SEQ ID
(SEQ ID NO: 133)




NO: 32)



E8H11
QVQLVESGGGVVQPGRSLRLSCAASGFTFS
SYGMH
WVRQAPGKGLEWVA



(SEQ ID NO: 131)
(SEQ ID
(SEQ ID NO: 7)




NO: 41)



E8H7
QVQLQESGGGLVQPGGSLRLSCSASGFTFS
SYAMH
WVRQAPGKGLEYVS



(SEQ ID NO: 180)
(SEQ ID
(SEQ ID NO: 181)




NO: 29)



E8G12
QVQLQQSGGGLVQPGGSLRLSCAASGFTFS
SYAMS
WVRQAPGKGLEWVS



(SEQ ID NO: 182)
(SEQ ID
(SEQ ID NO: 133)




NO: 32)



E8F11
QVQLQQSGGGLVQPGGSLRLSCAASGFTFS
SYAMS
WVRQAPVKGLEWVS



(SEQ ID NO: 183)
(SEQ ID
(SEQ ID NO: 184)




NO: 32)



E8C9
QVNLRESGGGLVKPGGSLRLSCAASGFTFS
SYAMS
WVRQAPGKGLEWVS



(SEQ ID NO: 185)
(SEQ ID
(SEQ ID NO: 133)




NO: 32)



D6G9
QVQLQESGGGLVQPGGSLRLSCAASGFTFS
SYAMG
WVRQAPGKGLEWVS



(SEQ ID NO: 74)
(SEQ ID
(SEQ ID NO: 133)




NO: 186)



D6D3
QVQLQESGGGLVQPGGSLRLSCAASGFTFS
SYAM(SEQ
WVRQAPGKGLEWVS



(SEQ ID NO: 5)
ID NO: 32)
(SEQ ID NO: 133)












clone
VH CDR2
VH FR3





F2-1A6
IIYPGDSDTRYSPSFQG (SEQ ID
QVTISADKSISTAYLQWSSLKASDTAMYYCAR



NO: 8)
(SEQ ID NO: 9)


F2-1H9
AVKQDGSEKYYLDSVKG (SEQ ID
RFTISRDNAKSSLYLQMDSLSVEDTAVYYCAR



NO: 10)
(SEQ ID NO: 11)


D1C5
VISYDGSNKYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 45)
(SEQ ID NO: 50)


D1D1
AI SADGAGTYYGDSVKG
RFTVSRDNFKSTLYLQMNRLRAEDTAVYYCAK



(SEQ ID NO: 82)
(SEQ ID NO: 139)


HB8
AITGGGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTALYYCAK



(SEQ ID NO: 140)
(SEQ ID NO: 141)


HC2
AISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 142)
(SEQ ID NO: 50)


HC4
AISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 142)
(SEQ ID NO: 46)


HE3
AISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 142)
(SEQ ID NO: 50)


HF1
AISGSGSGTYYADSVKG
RFTISRDNSKNTLYLQMNTLRAEDTALYYCAK



(SEQ ID NO: 143)
(SEQ ID NO: 144)


HH3
AITSSGGRTYYADSVRG
RLTISRDNSKNTLYLQMNTLRAEDTAVYYCAK



(SEQ ID NO: 145)
(SEQ ID NO: 146)


E8H11
VISYDGSNKYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 45)
(SEQ ID NO: 50)


E8H7
AISSNGGSTYYADSVKG
RFTISRDNSKNTLYLQMSSLRAEDTAVYYCVA



(SEQ ID NO: 187)
(SEQ ID NO: 188)


H8G12
AISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 142)
(SEQ ID NO: 50)


E8F11
AISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRIEDTAVYYCGR



(SEQ ID NO: 142)
(SEQ ID NO: 189)


E8C9
VIS-DGSTTYYADSVKG
RFTISRDNSKNMLYLQTNSLRAEDTAVYYCAK



(SEQ ID NO: 190)
(SEQ ID NO: 191)


D6G9
AISASGGSTYYADSVKG
RFTISRDNSKNTLYLQLNSLRAEDTAVYYCAK



(SEQ ID NO: 192)
(SEQ ID NO: 193)


D6D3
AITSSGGRTYYADSVRG
RLTISRDNSKNTLYLQMNTLRAEDTAVYYCAK



(SEQ ID NO: 145)
(SEQ ID NO: 146)





clone
VH CDR3
VH FR4





F2-1A6
RLHGPFYFDY (SEQ ID NO: 12)
WGQGTLVTVSS (SEQ ID NO: 13)


F2-1H9
GLRT (SEQ ID NO: 14)
MGQGTLVTVSS (SEQ ID NO: 15)


D1C5
GYGSGSYWSGAFDI
WGQGTMVTVSS



(SEQ ID NO: 147)
(SEQ ID NO: 43)


D1D1
LGGESYSAD
WGQGTLVTVSS



(SEQ ID NO: 148)
(SEQ ID NO: 13)


HB8
TLGRSTVATDY
WGQGTLVTVSS



(SEQ ID NO: 149)
(SEQ ID NO: 13)


HC2
GPGAAQDY
WGQGTLVTVSS



(SEQ ID NO: 150)
(SEQ ID NO: 13)


HC4
GAVGATTAFDY
WGQGTLVTVSS



(SEQ ID NO: 151)
(SEQ ID NO: 13)


HE3
VASSSSLGMDV
WGQGTTVTVSS



(SEQ ID NO: 152)
(SEQ ID NO: 153)


HF1
SVVGATSLDY
WGQGTLVTVSS



(SEQ ID NO: 154)
(SEQ ID NO: 13)


HH3
GIVGATAFDY
WGQGTLVTVSS



(SEQ ID NO: 155)
(SEQ ID NO: 13)


E8H11
DYGYCSGGSCYSPFDY
WGQGTLVTVSS



(SEQ ID NO: 194)
(SEQ ID NO: 13)


E8H7
RLEWL-----PLAWDY
WGQGTLVTVSS



(SEQ ID NO: 195)
(SEQ ID NO: 13)


E8G12
AARIAAR---PGPLDY
WGQGTLVTVSS



(SEQ ID NO: 196)
(SEQ ID NO: 13)


E8F11
H--LSSG----SSVDY
WGQGTLVTVSS



(SEQ ID NO: 197)
(SEQ ID NO: 13)


E8C9
AGPRTTV----TTVDS
WGQGTLVTVSS



(SEQ ID NO: 198)
(SEQ ID NO: 13)


D6G9
GLKDSSG------FDY
WGQGTLVTVSS



(SEQ ID NO: 199)
(SEQ ID NO: 13)


D6D3
GIVGATA------FDY
WGQGTLVTVSS



(SEQ ID NO: 155)
(SEQ ID NO: 13)
















TABLE 2





Light chain FRs and CDRs of antibodies that specifically bind CD44


















clone
VL FR1
VL CDR1
VL FR2





F2-
SELTQDPAVSVALGQTVRITC (SEQ
QGDSLRSYYAS (SEQ
WYQQKPGQAPLLVIY


1A6
ID NO: 16)
ID NO: 17)
(SEQ ID NO: 18)


F2-
SELTQDPAVSVALGQTVRITC (SEQ
QGDSLRSYYAS (SEQ
WYQRKPGQAPLLVIY


1H9
ID NO: 16)
ID NO: 17)
(SEQ ID NO: 19)


D1C5
EIVLTQSPLSLPVTPGEPASISC
RSSQSLLHTNGYNYLD
WYLQKPGQSPQLLIY



(SEQ ID NO: 156)
(SEQ ID NO: 157
(SEQ ID NO: 158)


D1D1
HVILTQDPAVSVALGQTVKITC
QGDSLRSYYAS
WYQQKPGQAPLLVLY



(SEQ ID NO: 159)
(SEQ ID NO: 17)
(SEQ ID NO: 160)


HB8
SELTQDPAVSVALGQTVRITC
QGDSLRSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


HC2
QSVLTQDPAVSVALGQTVTITC
QGDSLRSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 161)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


HC4
SELTQDPAVSVALGQTVKITC
QGDSLRSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 51)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


HE3
SELTQDPAVSVALGQTVRITC
QGDSLRSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


HF1
QSVLTQDPAVSVALGQTVRITC
QGDSLRDYYAS
WYKQKPGQAPLLVFF



(SEQ ID NO: 162)
(SEQ ID NO: 163)
(SEQ ID NO: 164)


HH3
SELTQDPAVSVALGQTVRITC
QGDSLRSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


E8H11
NFMLTQDPAVSVALGQTVRITC
QGDSLRS---YYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 200)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


E8H7
S-ELTQDPAVSVALGQTVRITC
QGDSLRS---YYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


E8G12
S-ELTQDPAVSVAVGQTVKITC
QGDSLRN---YYAS
WYQQKPRQAPVLVIY



(SEQ ID NO: 201)
(SEQ ID NO: 202)
(SEQ ID NO: 203)


E8F11
-SELTQPPSASGSPGQSVTISC
TGTSSDVGGYNYVS
WYQQRPGYAPKLMIY



(SEQ ID NO: 204)
(SEQ ID NO: 205)
(SEQ ID NO: 206)


E8C9
QSVLTQPPSVSGAPGQRVTISC
TGSSSNIGAGYDVH
WYQQLPGTAPKLLTY



(SEQ ID NO: 207)
(SEQ ID NO: 208)
(SEQ ID NO: 209)


D6G9
NFMLTQDPAVSVALGQTVRITC
QGDSLRS---YYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 200)
(SEQ ID NO: 17)
(SEQ ID NO: 70)


D6D3
S-ELTQDPAVSVALGQTVRITC
QGDSLRS---YYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 70)












clone
VL CDR2
VL FR3





F2-1A6
GKNIRPS (SEQ ID NO: 20)
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC




(SEQ ID NO: 23)


F2-1H9
GKNIRPS (SEQ ID NO: 20)
GIPDRFSGSSSGNTASLIITGAQAEDEADYYC




(SEQ ID NO: 22)


D1C5
LGSNRAS
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC



(SEQ ID NO: 165)
(SEQ ID NO: 166)


D1D1
GENNRPS
GIPDRFSGSGSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 71)
(SEQ ID NO: 167)


HB8
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


HC2
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


HC4
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


HE3
GKNNRPS
GIPDRFSGSSSGNTASLTITGARAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 169)


HF1
GKSNRPS
GIPNRFSGSTSGSTATLTVTGAQAEDEADYFC



(SEQ ID NO: 170)
(SEQ ID NO: 171)


HH3
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


E8H11
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


E8H7
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


E8G12
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


E8F11
DVSNRPS
GVSNRFSGSKSGNSASLDISGLQSEDEADYYC



(SEQ ID NO: 210)
(SEQ ID NO: 211)


E8C9
GNSNRPS
GVPDRFSGSKSGTSASLAITGLQAEDEADYYR



(SEQ ID NO: 212)
(SEQ ID NO: 213)


D6C9
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


D6D3
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)





clone
VL CDR3
VL FR4





F2-1A6
NSRDSSGNHVV (SEQ ID NO: 23)
FGGGTKLTVLG (SEQ ID NO: 27)


F2-1H9
NSRDSSANQM (SEQ ID NO: 25)
FGGGTKLTVLG (SEQ ID NO: 27)


D1C5
MQGRQSPLT
FGGGTKVEIKR



(SEQ ID NO: 172)
(SEQ ID NO: 84)


D1D1
HSRDSSGKYV
FGVGTKVTVLG



(SEQ ID NO: 173)
(SEQ ID NO: 174)


HB8
NSRDSSGNPHVV
FGGGTKLTVLG



(SEQ ID NO: 175)
(SEQ ID NO: 27)


HC2
NSRDSSGNHVV
FGGGTKVTVLG



(SEQ ID NO: 23)
(SEQ ID NO: 80)


HC4
NSRDSSGNHLV
FGGGTKLTVLG



(SEQ ID NO: 176)
(SEQ ID NO: 27)


HE3
TSRDSSGKQLV
FGGGTKLTVLG



(SEQ ID NO: 177)
(SEQ ID NO: 27)


HFI
SSRDSSGRLIL
FGGGTKLTVLG



(SEQ ID NO: 178)
(SEQ ID NO: 27)


HH3
NSRDSSGNSVV
FGGGTKVTVLG



(SEQ ID NO: 179)
(SEQ ID NO: 80)


E8H11
HSRDSSGN-YL
FGGGTKLTVLG



(SEQ ID NO: 214)
(SEQ ID NO: 27)


E8H7
NSRDSSGNHKV
FGGGTKLTVLG



(SEQ ID NO: 215)
(SEQ ID NO: 27)


E8G12
NSRDRSNNHLL
FGGGTKLTVLG



(SEQ ID NO: 216)
(SEQ ID NO: 27)


E8F11
AAWDDSLREFL
FGTGTKVTVLG



(SEQ ID NO: 217)
(SEQ ID NO: 218)


E8C9
SAWDSSLFNWV
FGGGTKLTVLG



(SEQ ID NO: 219)
(SEQ ID NO: 27)


D6G9
NSRDSSAKRVV
FGGGTKVTVLG



(SEQ ID NO: 220)
(SEQ ID NO: 80)


D6D3
NSRDSSGNSVV
FGGGTKVTVLG



(SEQ ID NO: 179)
(SEQ ID NO: 80)









Anti-EphA2
Antibodies of the present disclosure include those that specifically bind EphA2. Anti-EphA2 antibodies encompass those that competitively bind to an epitope (e.g. on the extracellular domain) of EphA2 with 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The epitope bound by anti-EphA2 antibodies reside in a contiguous amino acid sequence of EphA2 from residue position 25 to 534, as set forth below.







(SEQ ID NO: 26)


QGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMS


 


GDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYA


 


ESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLT


 


RKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLA


 


TVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQ


 


ACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMP


 


CTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESG


 


ECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTS


 


RSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYR


 


KKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEF


 


QTLSPEGSGN






The residue position numbers of EphA2 are determined based on the sequence set forth in GenBank Accession No. NP_004422.2 or UniProt Accession No. P29317.
Anti-EphA2 antibodies also encompass those that competitively bind to the ligand binding site of EphA2. For example, antibodies 2D6, D2-1A7, and D2-1A9 compete with Ephrin A1, the natural ligand of EphA2, for binding to EphA2. Thus, the subject antibodies can also compete with Ephrin A1 for binding to an epitope of EphA2.
Antigens that comprise epitopes that are similar to the epitopes of EphA2 can also be binding targets of subject antibodies. When bound to an antigen on a cell surface (e.g. EphA2), certain of the subject antibodies will be internalized by the cell.
Epitopes for which anti-EphA2 antibodies have affinity are cell-surface exposed and solvent-accessible on many cancer cells, particularly on the plasma membrane of cells. The epitopes can be accessible to the subject antibodies when the cells are live. The cancer cells can include those derived from tissue of epithelial in origin. For example, the epitopes can be found on cancer cells derived from breast cancers, skin cancers (e.g. melanoma), lung cancers, prostate cancers, colon cancers, and/or ovarian cancers. Other cancers cells for which anti-EphA2 antibodies have affinity may be liver cancer, esophageal squamous cell carcinoma, epidermoid cancer, pancreatic cancer, glioblastomas, neuroblastomas, and/or other neural cancers, for example.
Additional examples of subject antibodies encompass those that have the same binding specificities and contain at least two CDRs that each independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of a VH CDR of antibodies shown in FIGS. 8, 46, and 47, and in tables below (e.g. VH CDR1 of 2D6; or VH of CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15E11). The subject antibody can also include all three CDRs from any VH of any antibody shown in FIGS. 8, 46, and 47, such that each VH CDR in the subject antibody is selected from a single antibody shown in FIG. 8, 46, or 47 or tables below (e.g., Table 3) and each VH CDR independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of the corresponding VH CDR of an antibody shown in FIGS. 8, 46, and 47 or in tables below. For example, the heavy chain of a subject antibody can contain at least two VH CDRs or all three VH CDRs of 2D6. Alternatively, the heavy chain can contain at least two VH CDRs or all three VH CDRs of D2-1A7. Other examples include a heavy chain that contains at least two VH CDRs or all three VH CDRs of D2-1A9 and a heavy chain that contains at least two VH CDRs or all three VH CDRs of D2-1B1. As further examples, the heavy chain of a subject antibody can contain at least two VH CDRs or all three VH CDRs of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
Similarly for the light chain, a subject antibody can contain at least two CDRs that are each independently at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of a VL CDR of an antibody shown in FIG. 8, 46, or 47 or tables below (e.g. VL CDR1 of 2D6; or VL of CDR1 of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11). The subject antibody can also or alternatively include all three VL CDRs from any of antibodies shown in FIGS. 8, 46, and 47 or tables below (e.g., Table 4) and each VL CDR independently shares at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity with the amino acid sequence of a VL CDR of an antibody shown in FIG. 8, 46, or 47 or tables below. For example, the light chain of a subject antibody can contain two VL CDRs or all three VL CDRs of 2D6. Alternatively, the light chain can contain two VL CDRs or all three VL CDRs of D2-1A7. Other examples include a light chain that contains two VL CDRs or all three VL CDRs of D2-1A9 and a light chain that contains two VL CDRs or all three VL CDRs of D2-1B1. As further examples, the light chain of a subject antibody can contain two VL CDRs or all three VL CDRs of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
Optionally, antibodies can contain the exactly the same (i.e. 100% identity), similar, or different framework sequences (FR) in any of corresponding framework sequences in the heavy or light chain provided in FIGS. 8, 46, and 47 or tables below (e.g., Tables 3 and 4). Where the framework sequences are similar, the framework may be at least about 85%, at least about 86%, at least about 90%, at least about 93%, at least about 96%, at least about 98%, or up to 100% identity to a corresponding framework sequence in any of antibodies shown in FIGS. 8, 46, and 47 or tables below.
An antibody of the present disclosure may therefore contain a full-length VH and/or full length VL sequence that is at least 80% identity, at least 85%, at least 90%, at least 95%, up to 100% amino acid sequence identity to a full-length VH or VL sequence shown in FIG. 8, 46, or 47 or tables below. For example, a subject antibody can contain the full length VH and/or full length VL of 2D6. Alternatively, the subject antibody can contain the full length VH and/or full length VL of D2-1A7. Other examples include an antibody containing the full length VH and/or full length VL of D2-1A9 and an antibody containing the full length VH and/or full length VL of D2-1B1. Further examples include an antibody containing the full length VH and/or full length VL of A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. Examples of antibodies of the present disclosure that bind to EphA2 are listed in tables below.







TABLE 3





Heavy chain FRs and CDRs of antibodies that specifically bind


EphA2


















clone
VH FR1
VH CDR1
VH FR2





2D6
QVQLQESGGGLVQPGGSLRLSCAASGFTFS
SYAMS
WVRQAPGKGLDWVS



(SEQ ID NO: 5)
(SEQ ID
(SEQ ID NO: 53)




NO: 32)



D2-
QVQLQQSGGGVVQPGRSLRLSCAASGFTFS
SYAMH
WVRQAPGKGLEWVA


1A7
(SEQ ID NO: 28)
(SEQ ID
(SEQ ID NO: 7)




NO: 29)



D2-
QVQLVESGGGLIQPGGSLKLSCAASGFTVS
NSYMS
WVRQAPGKGLEWVA


1A9
(SEQ ID NO: 30)
(SEQ ID
(SEQ ID NO: 7)




NO: 31)



D2-
QVQLQESGGGVVQPGRSLRLSCAASGFTFS
SYGMH
WVRQAPGKGLEWVA


1B1
(SEQ ID NO: 54)
(SEQ ID
(SEQ ID NO: 7)




NO: 41)



A3H9
QVLLVESGGGVVQAGASLRVSCAASGFSLT
SYGMH
WVRQAPGKGLEWVA



(SEQ ID NO: 126)
(SEQ ID
(SEQ ID NO: 7)




NO: 41)



A3G3
QVQLVESGGGVVQPGRSLRLSCAASGFTFS
SYAMG
WVRQAPGKGLEWVA



(SEQ ID NO: 131)
(SEQ ID
(SEQ ID NO: 7)




NO: 186)



A3D10
LVQLVQSGGGLVKPGGSLRLSCAASGFTFS
TYSMN
WVRQAPGKGLEWVS



(SEQ ID NO: 434)
(SEQ ID
(SEQ ID NO: 133)




NO: 222)



A3D1
QVQLVQSGGGLVQPGGSLRLSCAASGFTFS
SYGMH
WVRQAPGKGLEWVA



(SEQ ID NO: 182)
(SEQ ID
(SEQ ID NO: 7)




NO: 41)



A3C8
QLQLVESGGGLVQPGGSLRLSCAASGFTFS
SYAMN
WVRQAPGKGLEWVS



(SEQ ID NO: 223)
(SEQ ID
(SEQ ID NO: 133)




NO: 224)



1A3
QVQLQQSGPGLVKPSQTLSLTCAISGDSVS
SNSAA
WNWIRQSPSRGLEWLG



(SEQ ID NO: 225)
(SEQ ID
(SEQ ID NO: 227)




NO: 226)



1A5
QVQLQESGGGVVQPGGSLRLSCAASGFTFS
NYAMH
WVRQAPGKGLEYVS



(SEQ ID NO: 228)
(SEQ ID
(SEQ ID NO: 181)




NO: 229)



1A8
QVQLQQSGGGLVQPGGSLRLSCSASGFTFS
NYAIH
WVRQAPGKGLEYVS



(SEQ ID NO: 230)
(SEQ ID
(SEQ ID NO: 181)




NO: 231)



1A12
QVQLQESGGGLVQPGGFLRLSCAASGFTFS
SYGMH
WVRQTPGKGLEWVS



(SEQ ID NO: 232)
(SEQ ID
(SEQ ID NO: 233)




NO: 41)



1B2
QVQLVESGGGVVQPGRSLRLSCAASGFTFS
SYGMH
WVRQAPGKGLEWVA



(SEQ ID NO: 131)
(SEQ ID
(SEQ ID NO: 7)




NO: 41)



1C2
QVQLQESGGGLVKPGGSLRLSCAGSGFIFN
TYSMN
WVRQSPGKGLEWVS



(SEQ ID NO: 234)
(SEQ ID
(SEQ ID NO: 235)




NO: 222)



1C7
QVQLVESGGGVVQPGRSLRLSCAASGFTFS
SYGMH
WVRQAPGKGLEWVS



(SEQ ID NO: 131)
(SEQ ID
(SEQ ID NO: 133)




NO: 41)



1D8
QVQLVESGGGLIQPGGSLRLSCAASGFTVS
SNYMS
WVRQAPGKGLEWVS



(SEQ ID NO: 236)
(SEQ ID
(SEQ ID NO: 133)




NO: 237)












clone
VH CDR2
VH FR3





2D6
IIYNGDNTYYADSVKG (SEQ ID
RFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR



NO: 34)
(SEQ ID NO: 35)


D2-
VISYDGSNKYYADSVKG (SEQ ID
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR


1A7
NO: 45)
(SEQ ID NO: 46)


D2-
VIYSAGNTYYADSVKG (SEQ ID
RFTISRDTSNNTVHLQMNSLRPEDTAVYYCAR


1A9
NO: 38)
(SEQ ID NO: 39)


D2-
VISYDGSNKYYADSVKG (SEQ ID
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK


1B1
NO: 45)
(SEQ ID NO: 50)


A3H9
FISSDGSDKYYVDSVKG
RFTISRDTSKNMMYLQMNSLTTEDTAVYYCAK



(SEQ ID NO: 238)
(SEQ ID NO: 239)


A3G3
VIYRDGHGYYADSVKG
RFTVSRDSSENTVYLQMNSLRAEDTAIYYCAS



(SEQ ID NO: 240)
(SEQ ID NO: 241)


A3D10
SISSSSSYIYYADSVKG
RFTISRDNANNSLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 242)
(SEQ ID NO: 243)


A3D1
VIWYDGSNKYYADSVKG
RFTISRDNSRNTLYLEMNSLRAEDTAVYYCVK



(SEQ ID NO: 244)
(SEQ ID NO: 245)


A3C8
AISGSGGNTYYADSVKG
RFTISRDNSNNALYLQMNSLRVEDTAVYYCAR



(SEQ ID NO: 246)
(SEQ ID NO: 247)


1A3
RTYYRSKWYNDYAVSVKS
RITINPDTSKNLFSLQLNSVTPEDTALYYCAR



(SEQ ID NO: 248)
(SEQ ID NO: 249)


1A5
SISSNGGGTYYADSVKG
RFTISRDDAKNTLYLQLNSLRDEDTAVYYCAK



(SEQ ID NO: 250)
(SEQ ID NO: 251)


1A8
AINSNGGSTYYADSVKG
RSIISRDNSMNTVYLQMSSLRAEDTAVYYCVK



(SEQ ID NO: 252)
(SEQ ID NO: 253)


1A12
YISSSSSYTNYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 254)
(SEQ ID NO: 46)


1B2
VISYDGSNKYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 45)
(SEQ ID NO: 46)


1C2
STGGSGKNTFYADSVRG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 255)
(SEQ ID NO: 256)


1C7
YISSSGSYTNYADSVKG
RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAK



(SEQ ID NO: 257)
(SEQ ID NO: 258)


1D8
VIYSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR



(SEQ ID NO: 259)
(SEQ ID NO: 46)





clone
VH CDR3
VH FR4





2D6
WSGTSYDY (SEQ ID NO: 62)
WGQGTLVTVSS (SEQ ID NO: 13)


D2-
ASVGATGPFDI (SEQ ID NO: 42)
WGQGTMVTVSS (SEQ ID NO: 43)


1A7




D2-
EGSFGYDY (SEQ ID NO: 44)
RGQGTLVTVSS (SEQ ID NO: 75)


1A9




D2-
VIAGGAYYGSADY (SEQ ID NO: 76)
WGQGTLVTVSS (SEQ ID NO: 13)


1B1




A3H9
DWGSNWYLFDY
WGQGTLVTVSS



(SEQ ID NO: 260)
(SEQ ID NO: 13)


A3G3
HDYAGNPAGSASGY
WGQGTLVTVSS



(SEQ ID NO: 261)
(SEQ ID NO: 13)


A3D10
GNTVAQRLDVFDY
WGQGTLVTVSS



(SEQ ID NO: 262)
(SEQ ID NO: 13)


A3D1
DRQPDGRWPFDL
WGQGTLVTVSS



(SEQ ID NO: 263)
(SEQ ID NO: 13)


A3C8
DASYYADDY
WGQGTLVTVSS



(SEQ ID NO: 264)
(SEQ ID NO: 13)


1A3
EEDYSGFQH
WGQGTLVTVSS



(SEQ ID NO: 265)
(SEQ ID NO: 13)


1A5
DYFGSIDY
WGQGTLVTVSS



(SEQ ID NO: 266)
(SEQ ID NO: 13)


1A8
EENGSGFDS
WGQGTLVTVSS



(SEQ ID NO: 267)
(SEQ ID NO: 13)


1A12
DNWYFDL
WGRGTLVTVSS



(SEQ ID NO: 268)
(SEQ ID NO: 269)


1B2
ALYYDEALDY
WGQGTLVTVSS



(SEQ ID NO: 270)
(SEQ ID NO: 13)


1C2
EDSSGSFDY
WGQGTLVTVSS



(SEQ ID NO: 271)
(SEQ ID NO: 13)


1C7
VRGWDGDYLDY
WGQGTLVTVSS



(SEQ ID NO: 272)
(SEQ ID NO: 13)


1D8
GGFSGYDYFDY
WGQGTLVTVSS



(SEQ ID NO: 273)
(SEQ ID NO: 13)
















TABLE 4





Light chain FRs and CDRs of antibodies that specifically bind


EphA2


















clone
VL FR1
VL CDR1
VL FR2





2D6
SELTQDPAVSVALGQTVRITC (SEQ
QGDSLRSYYAS
WYQQKPGQAPLLVIY



ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 18)


D2-
SELTQDPAVSVALGQTVSITC (SEQ
QGDSLRSYYAS
WYQQKPGQAPLLVIY


1A7
ID NO: 47)
(SEQ ID NO: 17)
(SEQ ID NO: 18)


D2-
DIVMTQSPGTLSLSPGERATLSC
RASQSVSSSFLG
WYQQKPGQAPRLLIY


1A9
(SEQ ID NO: 48)
(SEQ ID NO:
(SEQ ID NO: 77)




49)



D2-
SELTQDPAVSVALGQTVKITC
QGDSLRTYYAS
WYQQKPGQAPVLVIY


1B1
(SEQ ID NO: 51)
(SEQ ID NO:
(SEQ ID NO: 70)




52)



A3H9
SVLTQPPSASETPGQRVTISC
SGSSSNIGANTVH
WYQQFPGTAPKLLIY



(SEQ ID NO: 291)
(SEQ ID
(SEQ ID NO: 293)




NO: 292)



A3G3
SALTQPASVSGSPGQSVTISC
TGTSSDVGGYDYVS
WYQQHPGKAPKLVMY



(SEQ ID NO: 294)
(SEQ ID
(SEQ ID NO: 296)




NO: 295)



A3D10
SVLTQPPSVSGAPGQRVTISC
TGGSSNVGAGFDVH
WYQQLPGTAPKLLIY



(SEQ ID NO: 297)
(SEQ ID
(SEQ ID NO: 299)




NO: 298)



A3D1
SALTQPASVSGSPGQSVTISC
TGANSDLGGYNYVS
WYQHHPAKAPKLIIY



(SEQ ID NO: 300)
(SEQ ID
(SEQ ID NO: 302)




NO: 301)



A3C8
SELTQDPAVSVALGQTVRITC
QGDSLKSYYAS
WYQQKPGQAPVLVIY



(SEQ ID NO: 16)
(SEQ ID
(SEQ ID NO: 70)




NO: 303)



1A3
QSALTQPASVSGSPGQSITISC
TGTSSDVGGYNYVS
WYQQHPGKAPKLMIY



(SEQ ID NO: 304)
(SEQ ID
(SEQ ID NO: 435)




NO: 205)



1A5
PELTQDPAVSVALGQTVTITC
QGDSLRSYYAS
WYQQKPGQAPLLVIY



(SEQ ID NO: 436)
(SEQ ID NO: 17)
(SEQ ID NO: 18)


1A8
EIVLTQSPSSVSASVGDRVTITC
RASQDISKWLA
WYQQRPGKVPRLLIY



(SEQ ID NO: 437)
(SEQ ID
(SEQ ID NO: 439)




NO: 438)



1A12
DVVMTQSPSTLSASVGDRVSITC
RASESISRWLA
WYQQKPGKAPKALIY



(SEQ ID NO: 440)
(SEQ ID
(SEQ ID NO: 442)




NO: 441)



1B2
EIVLTQSPSSLSASVGDRVTITC
RASQDMSRWLA
WYQQKPGKAPKLLIH



(SEQ ID NO: 443)
(SEQ ID
(SEQ ID NO: 445)




NO: 444)



1C2
DIQMTQSPSTLSASIGDRVTITC
RASEGIYHWLA
WYQQKPGKAPKLLIY



(SEQ ID NO: 446)
(SEQ ID
(SEQ ID NO: 448)




NO: 447)



1C7
DIQMTQSPSLLSASVGDRVTITC
RASQGINNYLA
WYQQKPGKAPKLLIY



(SEQ ID NO: 449)
(SEQ ID
(SEQ ID NO: 448)




NO: 221)



1D8
QSVLTQPPSVSGAPGQRVTISC
TGSSSNIGAGYDVH
WYQQLPGTAPKLLIY



(SEQ ID NO: 207)
(SEQ ID
(SEQ ID NO: 299)




NO: 208)












clone
VL CDR2
VL FR3





2D6
GENNRPS (SEQ ID NO: 71)
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC




(SEQ ID NO: 23)


D2-1A7
GENNRPS (SEQ ID NO: 71)
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC




(SEQ ID NO: 23)


D2-1A9
GASSRAT (SEQ ID NO: 55)
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC




(SEQ ID NO: 56)


D2-1B1
GENSRPS (SEQ ID NO: 57)
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC




(SEQ ID NO: 23)


A3H9
SYSQRPS
GVPDRFSDSKSGTSASLAISGLQSEDEADYYC



(SEQ ID NO: 395)
(SEQ ID NO: 396)


A3G3
SHNQRSS
GVPDRFSGSKSGNSASLDISGLQSEDEADYYR



(SEQ ID NO: 397)
(SEQ ID NO: 398)


A3D10
GDKNRPS
GVPDRFSGSRSGTSASLAITGLQAEDEADYYC



(SEQ ID NO: 399)
(SEQ ID NO: 400)


A3D1
EVNNRPS
GVSHRFSGSKSANTASLTISGLQAEDEADYYC



(SEQ ID NO: 401)
(SEQ ID NO: 402)


A3C8
GKNNRPS
GIPDRFSGSSSGNTASLTITGAQAEDEADYYC



(SEQ ID NO: 168)
(SEQ ID NO: 23)


1A3
EGSKRPS
GVPERFSGSNSGNTATLTIGRVEAGDEADYYC



(SEQ ID NO: 403)
(SEQ ID NO: 404)


1A5
GKNIRPS
GIPDRFSGSSSGNSASLTITGAQAEDEADYYC



(SEQ ID NO: 20)
(SEQ ID NO: 405)


1A8
SASSLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFASYFC



(SEQ ID NO: 406)
(SEQ ID NO: 407)


1A12
KASSLES
GVPSRFSGSGSATEFTLTINSLQPDDFATYYC



(SEQ ID NO: 408)
(SEQ ID NO: 409)


1B2
SASTLQS
GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC



(SEQ ID NO: 410)
(SEQ ID NO: 411)


1C2
KASSLAS
GAPSRFSGSGSGTDFTLTISSLQPDDFATYYC



(SEQ ID NO: 412)
(SEQ ID NO: 413)


1C7
AASTLQS
GVPSRFSGSGSGTEFTLTISGLQPEDFATYYC



(SEQ ID NO: 414)
(SEQ ID NO: 415)


1D8
VNSNRPS
GVPDRFSGSKSGTSASLAITGLQAEDEADYYC



(SEQ ID NO: 94)
(SEQ ID NO: 95)





clone
VL CDR3
VL FR4





2D6
HSRDSSGTHLRV (SEQ ID NO: 79)
FGGGTKVTVLG (SEQ ID NO: 80)


D2-1A7
NSRDSSGTHLTV (SEQ ID NO: 60)
FGGGTKLTVLG (SEQ ID NO: 27)


D2-1A9
QQYGISPLT (SEQ ID NO: 61)
FGGGTKVEIKR (SEQ ID NO: 84)


D2-1B1
HSRDSSGTHLRV (SEQ ID NO: 79)
FGGGTKLTVLG (SEQ ID NO: 27)


A3H9
AAWDDILNGWV
FGGGTKVTVLG



(SEQ ID NO: 274)
(SEQ ID NO: 80)


A3G3
AAWDDSLSEFL
FGTGTKLTVLG



(SEQ ID NO: 275)
(SEQ ID NO: 276)


A3D10
QAYDSSLRGSV
FGGGTKLTVLG



(SEQ ID NO: 277)
(SEQ ID NO: 27)


A3D1
SSYRSGGTYV
FGTGTKLTVLG



(SEQ ID NO: 278)
(SEQ ID NO: 276)


A3C8
HSRDSSGNHPVV
FGGGTKVTVLG



(SEQ ID NO: 279)
(SEQ ID NO: 80)


1A3
QAWDSTSDHVV
FGGGTKVTVLG



(SEQ ID NO: 280)
(SEQ ID NO: 80)


1A5
HSRDSSGTHLRV
FGGGTKLTVLG



(SEQ ID NO: 79)
(SEQ ID NO: 27)


1A8
QQASVFPVT
FGGGTKLEIKR



(SEQ ID NO: 281)
(SEQ ID NO: 282)


1A12
QQYSSYPLT
FGQGTKVDIKR



(SEQ ID NO: 283)
(SEQ ID NO: 284)


1B2
QQLGVYPLT
IGGGTKVEIKR



(SEQ ID NO: 285)
(SEQ ID NO: 286)


1C2
QQYHTISRT
FGPXTKLEIKR



(SEQ ID NO: 287)
(SEQ ID NO: 288)


1C7
QNLNSYPLT
FGGGTKVEIKR



(SEQ ID NO: 289)
(SEQ ID NO: 84)


1D8
QSYDSSLSGWV
FGGGTKLTVLG



(SEQ ID NO: 290)
(SEQ ID NO: 27)









It will be appreciated that the amino acid sequence of a CDR can also be defined using alternative systems, which will be readily apparent to and applied by the ordinarily skilled artisan (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1991 and Lefranc et al. IMGT, the international ImMunoGeneTics information system. Nucl. Acids Res., 2005, 33, D593-D597)). A detailed discussion of the IMGTS system, including how the IMGTS system was formulated and how it compares to other systems, is provided on the World Wide Web at www(dot)imgt.cines.fr/textes/IMGTScientificChart/Numbering/IMGTnumberingsTable.html. All amino acid sequences of CDR in the present disclosure are defined according to Kabat et al., supra, unless otherwise indicated.
The variable chains disclosed herein can be joined directly or through a linker (e.g., (Gly4Ser)3, SEQ ID NO: 81) to form a single-chain antibody. Details on linkers are discussed later below.
Method of Antibody Production
Using the information provided herein, the anti-CD44 and anti-EphA2 antibodies of the present disclosure are prepared using standard techniques well known to those of skill in the art.
For example, the nucleic acid sequences provided in FIG. 9 can be used to express the subject antibodies. Alternatively, the polypeptide sequences provided herein (see, e.g., tables above and/or FIGS. 7, 8, 9, and 44-47) can be used to determine appropriate nucleic acid sequences encoding the antibodies and the nucleic acids sequences then used to express one or more antibodies specific for EphA2 or CD44. The nucleic acid sequence(s) can be optimized to reflect particular codon “preferences” for various expression systems according to standard methods well known to those of skill in the art.
Using the sequence information provided, the nucleic acids may be synthesized according to a number of standard methods known to those of skill in the art. Oligonucleotide synthesis, is preferably carried out on commercially available solid phase oligonucleotide synthesis machines (Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) or manually synthesized using, for example, the solid phase phosphoramidite triester method described by Beaucage et. al. (1981) Tetrahedron Letts. 22(20): 1859-1862.
Once a nucleic acid encoding a subject antibody is synthesized it can be amplified and/or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to persons of skill in the art and are the subjects of numerous textbooks and laboratory manuals.
Expression of natural or synthetic nucleic acids encoding the antibodies of the present disclosure can be achieved by operably linking a nucleic acid encoding the antibody to a promoter (which is either constitutive or inducible), and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the antibody. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.
To obtain high levels of expression of a cloned nucleic acid it is common to construct expression plasmids which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing antibodies are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used.
The antibody gene(s) may also be subcloned into an expression vector that allows for the addition of a tag (e.g., a FLAG® tag, hexahistidine, and the like) at the C-terminal end or the N-terminal end of the antibody (e.g. scFv) to facilitate purification. Methods of transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with nucleic acids can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector. The culture of cells used in the present disclosure, including cell lines and cultured cells from tissue (e.g., tumor) or blood samples is well known in the art.
Once the nucleic acid encoding a subject antibody is isolated and cloned, one can express the nucleic acid in a variety of recombinantly engineered cells known to those of skill in the art. Examples of such cells include bacteria, yeast, filamentous fungi, insect (e.g. those employing baculoviral vectors), and mammalian cells.
Isolation and purification of a subject antibody can be accomplished according to methods known in the art. For example, a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and/or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification (or precipitation using Protein L or A), washing to remove non-specifically bound material, and eluting the specifically bound antibody. The isolated antibody can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the antibody may be isolated using metal chelate chromatography methods. Antibodies of the present disclosure may contain modifications to facilitate isolation, as discussed above.
The subject antibodies may be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The protein can present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified antibodies may be provided such that the antibody is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed proteins.
The antibodies produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. During purification from E. coli, for example, the expressed protein can be optionally denatured and then renatured. This can be accomplished, e.g., by solubilizing the bacterially produced antibodies in a chaotropic agent such as guanidine HCl. The antibody is then renatured, either by slow dialysis or by gel filtration. Alternatively, nucleic acid encoding the antibodies may be operably linked to a secretion signal sequence such as pelB so that the antibodies are secreted into the periplasm in correctly-folded form.
The present disclosure also provides cells that produce subject antibodies, where suitable cells include eukaryotic cells, e.g., mammalian cells. The cells can be a hybrid cell or “hybridoma” that is capable of reproducing antibodies in vitro (e.g. monoclonal antibodies, such as IgG). For example, the present disclosure provides a recombinant host cell (also referred to herein as a “genetically modified host cell”) that is genetically modified with one or more nucleic acids comprising nucleotide sequence encoding a subject antibody.
Techniques for creating recombinant DNA versions of the antigen-binding regions of antibody molecules which bypass the generation of hybridomas are also contemplated herein. DNA is cloned into a bacterial (e.g., bacteriophage), yeast (e.g. Saccharomyces or Pichia) insect or mammalian expression system, for example. One example of a suitable technique uses a bacteriophage lambda vector system having a leader sequence that causes the expressed antibody (e.g. Fab or scFv) to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. One can rapidly generate a great numbers of functional fragments (e.g. scFv) for those which bind the tumor associated antigen.
Modification
The present disclosure encompasses antibodies and nucleic acids that are modified to provide a desired feature, e.g., to facilitate delivery to a specific type of tissue and/or cells in a subject, to increase serum half-life, to supplement anti-cancer activity, etc. The antibodies of the present disclosure can be provided with or without modification, and include human antibodies humanized antibodies and chimeric antibodies. One way to modify a subject antibody is to conjugate (e.g. link) one or more additional elements at the N- and/or C-terminus or to any amino acid (including an internal amino acid) of the antibody, where such additional elements include, e.g., another protein and/or a drug or carrier molecule.
A subject antibody modified with one or more additional elements retains the desired binding specificity, while exploiting properties of the one or more additional elements to impart an additional desired characteristic. For example, a subject antibody can be conjugated to a second molecule that aids in solubility, storage or other handling properties, cell permeability, half-life, reduction in immunogenicity, controls release and/or distribution such as by targeting a particular cell (e.g., neurons, leucocytes, tumor cells, etc.) or cellular location (e.g., lysosome, endosome, mitochondria etc.), tissue or other bodily location (e.g., blood, neural tissue, particular organs etc.). Other examples include the conjugation of a dye, fluorophore or other detectable labels or reporter molecules for assays, tracking, imaging, and the like. More specifically, a subject antibody can be conjugated to a second molecule such as a peptide, polypeptide, dye, fluorophore, nucleic acid, carbohydrate, anti-cancer agent, lipid, radionuclide, and the like (e.g., at either the reducing or non-reducing end), such as the attachment of a lipid moiety, including N-fatty acyl groups such as N-lauroyl, N-oleoyl, fatty amines such as dodecyl amine, oleoyl amine, and the like.
For example, for antibodies that can be internalized into cells, the antibody or nucleic acids of the present disclosure may be further modified to increase or decrease the efficiency of delivery into cells. Gene delivery methods are also contemplated herein to deliver nucleic acids that direct expression of proteins (e.g., a subject antibody or other protein) in cells. The efficiency of cellular uptake (e.g. endocytosis) of antibodies can be increased or decreased by linking to peptides or proteins. For example, a given antibody can be linked to a ligand for a target receptor or large molecule that is more easily engulfed by endocytotic mechanisms, such as another antibody, where such an antibody is an “antibody conjugate.” The conjugate payload (e.g., a ligand) can also be released by acid hydrolysis or enzymatic activity when the endocytotic vesicle fuses with lysosomes. To modify cellular uptake, the conjugate can include a ligand that retains the antibody on the surface of a cell, which can be useful as a control for (e.g., to decrease) cellular uptake, or in some instances decrease uptake in one cell type while increasing it in others.
Where the antibody is linked to another antibody, the antibody may be bispecific. As noted above, bispecific antibodies refer to antibodies that are specific for two different epitopes that may or may not be of the same antigen.
Other features of a conjugated antibody may include one where the conjugate reduces toxicity relative to unconjugated antibody. Another feature is that the conjugate may target a type of cell or organ (e.g. cancerous cell or cancerous tissue) more efficiently than an unconjugated antibody.
Additional examples include an antibody conjugated with one or more molecules that complement, potentiate, enhance or can otherwise operate synergistically in connection with the antibody. The antibody can have attached an anti-cancer drug, e.g., for delivery to a site of a cancer to further facilitate cell killing or clearance, e.g., an anti-proliferation moiety (e.g., VEGF antagonist, e.g., an anti-VEGF antibody), a toxin (e.g., ricin, Pseudomonas exotoxin A, and the like), a radionuclide (e.g. 90Y, 131I, 177L, 10B for boron neutron capture, and the like), an anti-cancer agent, and/or an oligonucleotide (e.g. siRNA).
For example, an antibody may be formulated in a lipidic nanoparticle (e.g., a liposome) by covalent or non-covalent modifications. The antibody may be attached to the surface of a lipidic nanoparticle directly via an Fc region, for example. The antibody may also be covalently attached to a terminus of a polymer grafted at or inserted into the surface of a lipidic nanoparticle via a linker. Such conjugated lipidic nanoparticles may be referred to herein as “immunoliposomes”. The subject antibodies in an immunoliposome can act as a targeting moiety enabling the immunoliposomes to specifically bind to CD44 and/or EphA2 on the surface of cancer cells. The immunoliposomes can be loaded with one or more of the anti-cancer agents, such as small molecule, peptide, and/or nucleic acid (e.g. siRNAs). Methods of making and loading lipidic nanoparticles, such as liposomes and immunoliposomes, are known in the art, e.g. US 20100068255, US 20100008978, US 20090171077, US 20090155272, US 20070116753, US 20070110798, US 20070031484, US 20060147513, US 20050112065, US 20040037874, US 20040209366, US 20030003143, U.S. Pat. No. 7,135,177, U.S. Pat. No. 7,022,336, U.S. Pat. No. 6,803,053, U.S. Pat. No. 6,528,087, U.S. Pat. No. 6,214,388, U.S. Pat. No. 6,210,707, U.S. Pat. No. 6,110,491, U.S. Pat. No. 5,980,935, U.S. Pat. No. 5,380,531, U.S. Pat. No. 7,507,407, U.S. Pat. No. 7,479,276, and U.S. Pat. No. 7,462,603.
The antibodies of the present disclosure can optionally be modified to provide for improved pharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, and the like). Modifications that can enhance serum half-life are of interest. A subject antibody may be “PEGylated”, as containing one or more poly(ethylene glycol) (PEG) moieties. Methods and reagents suitable for PEGylation of a protein are well known in the art and may be found in U.S. Pat. No. 5,849,860. PEG suitable for conjugation to a protein is generally soluble in water at room temperature, and has the general formula R(O—CH2—CH2)nO—R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.
The PEG conjugated to the subject protein can be linear. The PEG conjugated to the subject protein may also be branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, “star-PEG's” and multi-armed PEG's such as those described in Shearwater Polymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” Star PEGs are described in the art including, e.g., in U.S. Pat. No. 6,046,305.
Where the subject antibody is to be isolated from a source, the subject protein can be conjugated to moieties the facilitate purification, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), a lectin, and the like. A subject protein can also be bound to (e.g., immobilized onto) a solid support, including, but not limited to, polystyrene plates or beads, magnetic beads, test strips, membranes, and the like.
Where the antibodies are to be detected in an assay, the subject proteins may also contain a detectable label, e.g., a radioisotope (e.g., 125I; 35S, and the like), an enzyme which generates a detectable product (e.g., luciferase, β-galactosidase, horse radish peroxidase, alkaline phosphatase, and the like), a fluorescent protein, a chromogenic protein, dye (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 152Eu, or others of the lanthanide series, attached to the protein through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin; fluorescent proteins; and the like. Indirect labels include antibodies specific for a subject protein, wherein the antibody may be detected via a secondary antibody; and members of specific binding pairs, e.g., biotin-avidin, and the like.
Any of the above elements that are used to modify the subject antibody may be linked to the antibody via a linker, e.g. a flexible linker. If present, the linker molecules are generally of sufficient length to permit the antibody and a linked carrier to allow some flexible movement between the antibody and the carrier. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
Where the linkers are peptide, the linkers can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
Flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 83) and GGGSn (SEQ ID NO: 87), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. Examples of flexible linkers include, but are not limited GGSG (SEQ ID NO:66), GGSGG (SEQ ID NO:67), GSGSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO: 88), GSSSG (SEQ ID NO: 89), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
Human Engineered Antibody
Where the antibodies of the present disclosure that binds CD44 or EphA2 are not human, the antibodies can be humanized. As used herein, a humanized antibody is a recombinant polypeptide that is derived from a non-human (e.g., rodent) antibody and has been modified to contain at least a portion of the framework and/or constant regions of a human antibody.
Humanized antibodies also encompass chimeric antibodies and CDR-grafted antibodies in which various regions may be derived from different species. Chimeric antibodies may be antibodies that include a variable region from any source linked to a human constant region. Thus, in chimeric antibodies, the variable region can be non-human, and the constant region is human. CDR-grafted antibodies are antibodies that include the CDRs from a non-human “donor” antibody linked to the framework region from a human “recipient” antibody. For example, an antibody of the present disclosure in a form of scFV may be linked to a human constant region (e.g. Fc region) to be made into a human immunoglobulin.
Fc Region
An antibody of the present disclosure that binds CD44 or EphA2 may contain an Fc region. The Fc region may be any of the naturally occurring isoforms found in human or other animals (e.g. derived from any classes or subclasses of immunoglobulins) and can optionally be further modified to have altered function. For example, the Fc region may be modified in one or more amino acid residue position to have increased effector functions, such as initiating cell-mediated cytotoxicity or activating complement activity (e.g. C1q binding or complement dependent cytotoxicity), downregulating cell-surface receptor, etc. Details of Fc variants that may be used as antibodies of the present disclosure may be found in, for example, U.S. Pat. No. 7,416,727, U.S. Pat. No. 7,371,826, U.S. Pat. No. 7,335,742, U.S. Pat. No. 7,355,008, U.S. Pat. No. 7,521,542, and U.S. Pat. No. 7,632,497.
Compositions
The subject compositions provide antibodies and/or nucleic acid encoding thereof, in which the antibodies bind to and are internalized by cancer cells. The compositions of the present disclosure find use in treating a subject (e.g., a human) containing cancer, and may be suitable for treatment during any stage of the disease. Compositions containing one, two, or more different antibodies can be provided as a pharmaceutical composition and administered to a mammal (e.g., to a human) in need thereof.
Compositions contemplated herein may contain one, two, three, or more different antibodies of the present disclosure (and/or nucleic acids encoding thereof). For example, the composition can contain one or more of the following: F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11. The composition may optionally further include antibodies containing one or more CDRs from these antibodies, and/or one or more antibodies containing mutants or derivatives of these antibodies.
An example of a composition of the present disclosure may include any of the combinations described above or one or more of the antibodies disclosed in FIG. 7, 8, or 9, or FIGS. 44-47. Where the composition contains two or more antibodies, each antibody can be specific to the same or different epitopes or to epitopes on different antigens. For example, the composition may contain at least one antibody specific for the epitope of CD44 or EphA2 and another antibody specific for another cell-surface antigen, such as EGFR. The composition may also contain dual-specific, polyspecific antibodies, or nucleic acids encoding thereof.
The antibodies of the present disclosure can be used individually, and/or in combination with each other (e.g. to form bispecific or polyspecific antibodies), and/or in combination with other known anti-cancer agents (e.g. antibodies for cancer treatment). For example, a composition, such as a liposome, can comprise two or more antibodies, in which at least one of the antibodies is an antibody of the present disclosure. As described above, the liposome may contain one or more antibodies that are different than the subject antibodies. Such liposome may be dual-specific, polyspecific, etc, so that the liposome is specific for an additional epitope (e.g. an epitope in EGFR or HER2) in addition to the epitope of the subject antibody.
Combinations can be provided in a single formulation or can be provided as separate formulations in a kit, where the separate formulations may contain a single antibody or two antibodies. Such separate formulations of a kit may be combined prior to administration or administered by separate injection.
A subject pharmaceutical composition can be provided in a pharmaceutically acceptable excipient, which can be a solution such as an aqueous solution, e.g., a saline solution, or can be provided in powder form. A subject composition may comprise other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
A subject antibody, e.g., in the form of a pharmaceutically acceptable salt, can be formulated for oral, topical or parenteral administration for use in the methods described later below. In certain embodiments, e.g., where an antibody is administered as a liquid injectable, e.g., suitable for intravenous injection) an antibody formulation may be a sterile, non-pyrogenic aqueous solution comprising salts (e.g., to adjust tonicity) buffers, preservatives, amino acids, and other pharmaceutically acceptable carriers and excipients, and may be provided as a ready-to-use dosage form, or as a reconstitutable storage-stable powder or liquid composed of pharmaceutically acceptable carriers and excipients. Formulation for convection enhanced delivery may be as described in, e.g., US 20090208422.
Compositions of the present disclosure can include a therapeutically effective amount of a subject antibody, as well as any other compatible components, as needed. By “therapeutically effective amount” is meant that the administration of that amount to an individual, either in a single dose, as part of a series of the same or different antibody or compositions, is effective to reduce the proliferation and/or metastases of a cancerous cell in a subject or to provide any other detectable therapeutic benefit. Such therapeutically effective amount of an antibody and its impact on cell growth includes cooperative and/or synergistic inhibition of cell growth in conjunction with one or more other therapies (e.g., immunotherapy, chemotherapy, radiation therapy etc.). As noted below, the therapeutically effective amount can be adjusted in connection with dosing regimen and diagnostic analysis of the subject's condition (e.g., monitoring for the presence or absence of a cell surface epitopes using an antibody specific for CD44 and/or EphA2) and the like.
Amount and Dosage
The exact dose will be ascertainable by one skilled in the art. The dosage can depend on a variety of factors including the strength of the particular compound employed, the condition of the subject, and the body weight of the subject, as well as the severity of the illness and the stage of the disease. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound. As known in the art, adjustments based on age, body weight, sex, diet, time of administration, drug interaction and severity of condition may be necessary and will be ascertainable with routine experimentation by those skilled in the art. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody fragment.
The amount of composition administered to a subject, e.g., a human, in the context of the present disclosure should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame, and varies depending upon the goal of the administration, the health and physical condition of the individual to be treated, age, the degree of resolution desired, the formulation of the antibody composition, the treating clinician's assessment of the medical situation, and other relevant factors. Thus it is expected that the amount will fall in a relatively broad range, but can nevertheless be routinely determined through various features of the subject such as note above.
As an example, a non-limiting range for a therapeutically or prophylactically effective amount of a subject antibody is from about 0.1 mg/kg to about 20 mg/kg, e.g., from about 1 mg/kg to about 10 mg/kg.
The concentration of an antibody in a pharmaceutical formulations can vary from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by consideration of fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected and the patient's needs. The resulting compositions may be in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like.
Also, suitable doses and dosage regimens can be determined by comparisons to anticancer or immunosuppressive agents that are known to affect the desired growth inhibitory or immunosuppressive response. Such dosages include dosages which result in the low dose inhibition of cell growth, without significant side effects. In proper doses and with suitable administration of certain compounds, the compounds of the present disclosure can provide for a wide range of intracellular effects, e.g., from partial inhibition to essentially complete inhibition of cell growth. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g., including ramp and maintenance doses). As indicated below, a subject composition may be administered in conjunction with other agents, and thus doses and regiments can vary in this context as well to suit the needs of the subject.
Combination Therapy
Any of a wide variety of cancer therapies can be combined in a composition with a subject antibody. For example, agents used in chemotherapeutic treatment or biological response modifier treatment may be present in the pharmaceutical composition comprising the antibody, such as an immunoliposome. Certain agents that can be used in combination with the subject antibodies are briefly discussed below.
Chemotherapeutic agents are non-proteinaceous compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (e.g., vinca) alkaloids, nucleic acids, such as inhibitory nucleic acids (e.g. siRNA), and steroid hormones.
Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, for example.
Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) can be used as anti-cancer agents. For example, taxanes, such as paclitaxel, as well as any active taxane derivative such as docetaxel, or a taxane pro-drug such as is 2′-(2-(N,N′-diethylamino)propionyl)-paclitaxel, 7-(2-(N,N′-diethylamino)propionyl)-paclitaxel, 2′-(2-(N,N′-diethylamino)propionyl)-docetaxel or 7-(2-(N,N′-diethylamino)propionyl)-docetaxel, are suitable.
Other anti-proliferative cytotoxic agents are navelbene, CPT-11 (irinotecan), anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. Microtubule affecting agents that have antiproliferative activity are also suitable for use. Hormone modulators and steroids (including synthetic analogs) are suitable for use.
Diagnostic Methods
The present disclosure provides a method of detecting a tumor associated antigen (e.g. CD44 or EphA2) in a biological sample in a subject or in a sample isolated from a subject. The methods are useful to both diagnostic and prognostic purposes. A subject method generally involves contacting a sample containing a cell with an antibody of the present disclosure; and detecting binding of the antibody to a cell in the sample. The cell can be in vitro, where the cell is in a biological sample obtained from a subject suspected for having cancer cells, a subject undergoing cancer treatment, or a subject being tested for susceptibility to treatment. The cell can be in vivo, e.g., the cell is in a subject suspected for having cancer cells, a subject undergoing treatment, or a subject being tested for susceptibility to treatment.
Antibodies can be used to detect cells expressing CD44 and/or EphA2 in a biological sample of a subject having or suspected of having cancerous cells via immunodiagnostic techniques. Such diagnostics can be useful to identify patients amenable to the therapies disclosed later below, and/or to monitor response to therapy.
Suitable immunodiagnostic techniques include, but are not necessarily limited to, both in vitro and in vivo (imaging) methods. For example, anti-CD44 or anti-EphA2 antibodies can be detectably labeled, administered to a subject suspected of having a cancer characterized by cell surface expression of CD44 or EphA2, and bound detectably labeled antibody detected using imaging methods available in the art.
The phrase “in vivo imaging” as used herein refers to methods of detecting the presence of an antibody (e.g. detectably labeled 2D6) in whole, live mammal. Optically detectable proteins such as fluorescent antibodies and luciferases-conjugated antibodies may be detected by in vivo imaging. Methods for using luciferases for real-time imaging of luciferase expression in live animals can be readily adapted for use in the subject methods disclosed herein (e.g., Greer L F et al., Luminescence 2002, 17: 43-74). In vivo imaging of fluorescent proteins in live animals is described in, e.g., Hoffman, Cell Death and Differentiation 2002, 9:786-789. In vivo imaging may be used to provide 2-D as well as 3-D images of a mammal. Radiolabeled antibodies, for example, may be administered to a subject and the subject imaged with a gamma camera. Charge-coupled device cameras, CMOS, or 3D tomographers may used to carry out in vivo imaging. For example, Burdette J E Journal of Mol. Endocrin., 40: 253-261, 2008, reviews utilizing computed tomography, magnetic resonance imaging, ultrasonography, positron emission tomography, single-photon emission computed tomography (SPECT), etc. The information from many in vivo imaging methods as those described above can provide information on cancer cells in the subject.
Where the methods are in vitro, the biological sample can be any sample in which a cancer cell may be present, including but not limited to, blood samples (including whole blood, serum, etc.), tissues, whole cells (e.g., intact cells), and tissue or cell extracts. For example, the assay can involve detection of CD44 and/or EphA2 on live cells or cells in a histological tissue sample. Particularly, detection can be assessed on an extracellular surface of a living cell. For example, the tissue sample may be fixed (e.g., by formalin treatment) and may be provided embedded in a support (e.g., in paraffin) or frozen unfixed tissue.
Assays can take a wide variety of forms, such as competition, direct reaction, or sandwich type assays. Examples of assays include Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as enzyme-linked immunosorbent assays (ELISAs); biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, and the like. The reactions generally include detectable labels conjugated to the antibody. Labels include those that are fluorescent, chemiluminescent, radioactive, enzymatic and/or dye molecules, or other methods for detecting the formation of a complex between antigen in the sample and the antibody or antibodies reacted therewith.
Where a solid support is used, the solid support is usually first reacted with a solid phase component under suitable binding conditions such that the antibody is sufficiently immobilized to the support. Sometimes, immobilization to the support can be enhanced by first coupling the antibody to a protein with better binding properties, or that provides for immobilization of the antibody on the support with out significant loss of antibody binding activity or specificity. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind antibodies to a support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like, with the proviso that the molecule used to immobilize the antibody does not adversely impact the ability of the antibody to specifically bind antigen. Such molecules and methods of coupling these molecules to the antibodies, are well known to those of ordinary skill in the art.
An ELISA method can be used, wherein the wells of a microtiter plate are coated with a subject antibody. A biological sample containing or suspected of containing CD44 and/or EphA2, is then added to the coated wells. After a period of incubation sufficient to allow antibody binding, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured antigen, the plate washed and the presence or absence of the secondary binding molecule detected using methods well known in the art.
Where desired, the presence or absence of bound CD44 and/or EphA2 from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. For example, a number of anti-bovine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal. In other related embodiments, competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.
Assays can also be conducted in solution, such that the antibodies and the antigens form complexes under precipitating conditions. An antibody-coated particle can be contacted under suitable binding conditions with a biological sample suspected of containing the target antigen to provide for formation of particle-antibody-antigen complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
Alternatively, assays for cellular uptake in live cells can be another diagnostic technique to positively identify cancerous cells. Since the subject antibodies are specifically internalized by cells expressing CD44 and/or EphA2, the cells can be allowed for internalization of the antibodies and any antibodies that are not internalized be washed away (e.g. acid wash). The internalized antibodies may be detected via its label as contained with the cells (e.g. FACS, spectrometer, radioisotope counter, etc.). Internalizing antibodies may also be selected for as described in U.S. Pat. No. 7,045,283.
The diagnostic assays described herein can be used to determine whether a subject has a cancer that is more or less amenable to therapy using antibody-based therapy, as well as monitor the progress of treatment in a subject. It also may be used to assess the course of other combination therapies. Thus, the diagnostic assays can inform selection of therapy and treatment regimen by a clinician.
The above-described assay reagents, including the antibodies of the present disclosure, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
Therapeutic Methods
A subject antibody finds therapeutic use in a variety of cancers. Subjects having, suspected of having, or at risk of developing cancer are contemplated for therapy and diagnosis described herein.
By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease, e.g., so as to decrease tumor load, which decrease can include elimination of detectable cancerous cells (e.g. metastatic cancer cells); and/or (iii) relief, that is, causing the regression of clinical symptoms.
A variety of subjects are treatable according to the methods. Generally such subjects are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the subjects will be humans.
In a related embodiment, the subject being treated possesses cells that express (e.g. overexpresses) a tumor associated antigen, CD44 and/or EphA2. The antigen is expressed on the cancer cell surface and is often present at a higher level than a corresponding non-cancerous cell. This aspect can be beneficial in the context of the methods of the present disclosure in that cells expressing or presenting CD44 and/or EphA2 can be amenable to treatment with an antibody of the present disclosure. The antibody can be administered to a subject, for example, where therapy is initiated at a point where presence of the antigen is not detectable, and thus is not intended to be limiting. It is also possible to initiate antibody therapy prior to the first sign of disease symptoms, at the first sign of possible disease, or prior to or after diagnosis of a disease.
Prodrugs of the antibody composition of the present disclosure are also contemplated in the methods described herein. Such prodrugs are in general functional derivatives of the compounds that are readily convertible in vivo into the required compounds. Thus, in the methods of the present disclosure, the term “administering” encompasses administering the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject in need thereof. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, e.g., in Wermuth, “Designing Prodrugs and Bioprecursors” in Wermuth, ed. The Practice of Medicinal Chemistry, 2d Ed., pp. 561-586 (Academic Press 2003).
Cancer
The antibody compositions may be advantageously used in an anti-cancer therapy, particularly where the cancerous cells present EphA2 and/or CD44 on an extracellularly accessible cell surface. One example is a cancer that presents an epitope bound by F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, HH3, 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11.
Antibody compositions described herein can be administered to a subject (e.g. a human patient) to reduce proliferation of cancerous cells, e.g., to reduce tumor size, reduce cancer load, reduce metastasis, and/or improve the clinical outcome in patients. For example, antibody compositions can be used to disrupt the cell cycle of the cancer cell, and facilitate entry of the cell into apoptosis. The methods relating to cancer contemplated herein include, for example, use of antibody therapy alone or in combination with anti-cancer vaccine or therapy.
Cancers particularly amenable to antibody therapy can be identified by methods similar to the diagnostic methods described above and others known in the art.
Types of Cancer
Where the anti-cancer therapy comprises administration of an antibody composition described previously, the anti-cancer therapy can be particularly directed to cancerous cells expressing cell-surface accessible and/or solvent-exposed epitopes bound by the subject antibodies, including metastatic cancer.
Examples of cancers presenting epitopes of CD44 that can be treated by the subject methods include cancers of the breast (e.g. basal cell breast cancer), colon, prostate, pancreas, etc. as well as adenoma and head and neck squamous cell carcinoma (HNSCC). Other cancers amenable to treatment can also be any cancer that is metastic and/or has metastatic potential.
Examples of cancers presenting epitopes of EphA2 include but not limited to cancer cells of epithelial origin. For example, the cancer cells can be derived from breast (e.g. basal cell breast cancer), skin (e.g. melanoma), lung, prostate, colon, and/or ovary. Other cancers cells for which anti-EphA2 antibodies have affinity may be liver cancer, esophageal squamous cell carcinoma, epidermoid cancer, pancreatic cancer, glioblastoma, neuroblastomas, and/or other neural cancers, for example.
It should be noted that while EphA2 or CD44 may be expressed at higher levels on a cancer cell compared to a non-cancerous cell, this is not a limitation of the therapies disclosed herein.
Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelieal carcinoma, and nasopharyngeal carcinoma.
Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
Other solid tumors that can be amenable to therapy by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Leukemias that can be amenable to therapy by a method disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like.
Other cancers that can be amenable to treatment according to the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinum.
Other examples of cancers that can be amenable to treatment using a methods disclosed herein include, but are not limited to, cancers of epithelial and neuroectodermal origin. Examples of epithelial origin include, but are not limited to, small cell lung cancer, cancers of the breast, eye lens, colon, pancreas, kidney, liver, ovary, and bronchial epithelium. The methods of the present disclosure may be used to treat cancer cells known to overexpress CD44 and/or EphA2.
Examples of cancers of neuroectodermal origin include, but are not limited to, Ewings sarcoma, spinal tumors, brain tumors, supratenbrial primative neuroectodermal tumors of infancy, tubulocystic carcinoma, mucinous tubular and spindle cell carcinoma, renal tumors, mediastinum tumors, neurogliomas, neuroblastomas, and sarcomas in adolescents and young adults.
Combinations with Other Cancer Therapies
As noted above, another feature of the methods is that an antibody can be administered to the subject in combination with one or more other therapies. Such therapy may be combined in a composition or be conjugated to the subject antibodies. In addition to being physically combined with antibodies disclosed herein (e.g, as a conjugate or in a liposome or other lipidic nanoparticle), one or more anti-cancer agents, may be administered in conjunction with, either simultaneously or before or after, administration of an antibody disclosed herein.
A therapy or treatment other than administration of antibody composition can be administered anywhere from simultaneously, to up to 5 hours or more, e.g., 10 hours, 15 hours, 20 hours or more, prior to or after administration of a subject antibody. A subject antibody and other therapeutic intervention are administered or applied sequentially, e.g., where a subject antibody is administered before or after another therapeutic treatment. A subject antibody and other therapy are administered simultaneously, e.g., where a subject antibody and a second therapy are administered at the same time, e.g., when the second therapy is a drug it can be administered along with a subject antibody as two separate formulations or combined into a single composition that is administered to the subject. Regardless of whether administered sequentially or simultaneously, as illustrated above, the treatments are considered to be administered together or in combination for purposes of the present disclosure.
Additional standard anti-cancer therapeutics that may or may not be administered in conjunction with a subject antibody, include but not limited to immunotherapy, chemotherapeutic agents and surgery (e.g., as those described further below). In addition, therapeutic administration of a subject antibody can also be post-therapeutic treatment of the subject with an anti-cancer therapy, where the anti-cancer therapy can be, for example, surgery, radiation therapy, administration of chemotherapeutic agents, and the like. Antibodies other than those disclosed herein, particularly monoclonal antibodies that can provide for complement-mediated killing, and/or antibody-dependent cellular cytotoxicity-mediated killing, of a target cell may also be used.
For example, a subject antibody can be administered in combination with one or more chemotherapeutic agents (e.g., cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP)), and/or in combination with radiation treatment and/or in combination with surgical intervention (e.g., pre- or post-surgery to remove a tumor), radiation therapy, bone marrow transplantation, biological response modifier treatment, and certain combinations of the foregoing. Radiation therapy includes, but is not limited to, X-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.
Routes of Administration
In practicing the methods, routes of administration (path by which a subject antibody is brought into a subject) may vary, where representative routes of administration for a subject antibody are described in greater detail below. A subject antibody alone or in combinations described above can be administered systemically (e.g., by parenteral, intravenous, intramuscular, intrathecal, intraventricular, or subcutaneous administration) or locally (e.g., at a local tumor site, e.g., by intratumoral administration (e.g., into a solid tumor, into an involved lymph node in a lymphoma or leukemia, or by convection enhanced delivery, e.g. into the brain, e.g., as disclosed in US 20090209937), administration into a blood vessel supplying a solid tumor, etc.), into a body cavity or lumen, or into an organ. These different routes of administration may be carried out by injection or infusion.
Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
The formulations of the present disclosure can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.
Formulations suitable for topical administration may be presented as transdermal compositions or transdermal delivery devices (“patches”), creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
Suppository formulations are also provided by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing the antibody compositions. Similarly, unit dosage forms for injection or intravenous administration may comprise the antibody in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Administration of the therapy can be repeated over a desired period, e.g., repeated over a period of about 1 day to about 5 days or once every several days, for example, about five days, over about 1 month, about 2 months, etc. It also can be administered prior, at the time of, or after other therapeutic interventions, such as surgical intervention to remove cancerous cells. The antibody can also be administered as part of a combination therapy, in which at least one of an immunotherapy, a cancer chemotherapy or a radiation therapy is administered to the subject (as described in greater detail above).
Screening Methods
The present disclosure also provides methods to screen for antibodies specific for antigens expressed on the cell surface (e.g. tumor associated antigens), as well as for internalization into mammalian cells upon binding to cell surface antigen. Some such methods are well known, see, e.g., U.S. Pat. No. 6,794,128 and U.S. Pat. No. 7,045,283. In one embodiment, methods are disclosed for screening antibody libraries (e.g., phage display libraries) for antibodies that bind to particular antigen(s) and are internalized into cells upon antigen binding. The antibody may be selected for its binding to a TAA (e.g. CD44 and/or EphA2) of interest and/or to a cancer cell, for example. The methods comprise initial internalizing antibody selection by several iterated rounds of library screening comprising selection for internalization on at least one mammalian cell line. Next, the collection of phage that express internalizing antibodies was selected against one or more antigen(s) (e.g., yeast displayed antigen, e.g., antigen known to be associated with a mammalian cell type) to isolate phage displaying antibodies against the desired antigen(s). The method may be executed according to the phage display, yeast display, and internalization selection methods described in the examples below.
Briefly, non-immune human scFv phage library is optionally depleted with control cells that do not express (e.g., do not appreciably express) the antigen(s) to be selected against to get rid of antibodies that may be nonspecifically interacting with cancer cells. The optionally depleted library is incubated with the live cancer cells of interest. The cancer cells may be derived from a known source or an unknown source. The cancer cells can also be derived exclusively from one cell line or one tumor or from a plurality of different cell lines or a plurality of different tumors. The process selects for antibodies that are internalized into cells by allowing the cells to endocytose the antibodies and stripping the cells of surface bound antibodies before proceeding to recover the phage from the cells. During panning and selection, the internalized phage are recovered from cell lysates and amplified. Multiple successive selection rounds (e.g. two or more) ensure selection of phage displaying a polypeptide that acts as a specific internalizing antibody for the cancer of interest.
Optionally before incubation with the yeast library, the selected phage may be depleted with control yeasts (e.g. yeasts that are not expressing an antigen of interest and/or expressing an irrelevant protein). The phage previously selected for cancer cell internalization are then incubated with yeast displaying one or more TAAs of interest (e.g. an extracellular domain of EphA2). The yeast incubated with the phage for further selection can express a plurality of TAAs, each being a different peptide fragment of the same full-length protein and/or each derived from different TAA. Where the yeast display a plurality of TAA, the antibodies specific for different TAA may be selected in parallel. The TAA may also encompass known tumor associated antigen and/or antigen whose cancer association is yet to be verified (e.g. suspected of being associated with cancer). After incubation, the phage bound to yeast displaying TAA of interest is eluted. Multiple successive rounds (e.g. two or more) may be carried out against the yeast library.
The stringency of the selection against yeast library can increase over each successive round (e.g. two or more). Many techniques well known in the art may be employed to increase the specificity of the recovered phage. Examples include increased wash times, increased detergent concentrations, increased salt concentrations, and inclusion of known macromolecular inhibitors (e.g. BPTI, Ecotin, and/or previously identified antibody inhibitors). Characterization of antibodies may include ELISAs and inhibition assays. Details on the assays to be performed in the method for selecting and isolating a polypeptide that can act as an anti-TAA agent are known in the art.
Compared to the use of phage to display antigens, the use of a simple eukaryote such as yeast for antigen display can result in a greater proportion of antigens being displayed in their proper conformation.
Kits and Systems
Also provided are kits and systems that find use in practicing the methods, as described above. For example, kits and systems may include one or more of the compositions described herein, such as an anti-CD44 and/or anti-EphA2 antibody (e.g. 2D6, D2-1A7, D2-1A9, D2-1B1, A3H9, A3G3, A3D10, A3D1, A3C8, 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, 1D8, or 15H11; or F2-1A6, F2-1H9, E8H11, E8H7, E8G12, E8F11, E8C9, D6G9, D6D3, D1C5, D1D1, HB8, HC2, HC4, HE3, HF1, or HH3), a nucleic acid encoding the same (especially a nucleic acid encoding a CDR of a heavy and/or light chain of any subject antibodies described above), or a cell containing the same. Other optional components of the kit include: buffers, etc., for administering the subject antibody, and/or for performing a diagnostic assay. The recombinant nucleic acids of the kit may also have restrictions sites, multiple cloning sites, primer sites, etc to facilitate their ligation to constant regions of nucleic acids. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.
The kits and systems for practicing the methods may include one or more pharmaceutical formulations that include the antibody compositions described herein. As such, the kits may include a single pharmaceutical composition present as one or more unit dosages. The kits may also include two or more separate pharmaceutical compositions.
In addition to the above components, the kits may further include instructions for practicing the methods. These instructions may be present in the kits in a variety of forms, one or more of which may be present in or on the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in or on the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
A kit may be provided for use in treating a host suffering from a cellular proliferative disease. This kit includes a pharmaceutical composition comprising antibody specific for EphA2 or CD44, and instructions for the effective use of the pharmaceutical composition in a method of treating a host suffering from a cancerous condition by inhibiting the growth of a cancer cell in a subject. Such instructions may include not only the appropriate handling properties, dosing regiment and method of administration, and the like, but can further include instructions to optionally screen the subject for a CD44- and/or EphA2-associated disease. This aspect can assist the practitioner of the kit in gauging the potential responsiveness of the subject to treatment with an antibody of the present disclosure, including timing and duration of treatment relative to the type and growth stage of the cancer. Thus in another embodiment, the kit may further include an antibody or other reagent for detecting an epitope of EphA2 or CD44 on an extracellularly accessible surface of a cancer cell. In another embodiment, the kit includes antibody that comprises a conjugate with a detectable label, such as a fluorophore.
The term “system” as employed herein refers to a collection of antibodies described herein and one or more second therapeutic agents, present in single or disparate compositions that are brought together for the purpose of practicing the methods. For example, separately obtained antibody specific to a TAA and chemotherapy dosage forms brought together and coadministered to a subject are a system according to the present disclosure.
EXAMPLES
The following examples are offered to illustrate, but not to limit any embodiments provided by the present disclosure.
The following methods and materials were used in the present example.
Methods and Materials
Cell Lines, Media, Antibodies and Full-Length cDNA Clones.
Breast cancer cell lines MCF7, T47D, MDAMB453, MDAMB231, human mammary epithelial cell (HMEC), and SUM52PE were obtained from the ATCC and Clontech (HMEC), or from collections developed in the laboratories of Dr. Steve Ethier (SUM52PE). The cell lines were cultured using conditions described previously (Neve R M et al. (2006) Cancer Cell 10:515-27). Yeast strain EBY100 was grown in YPD medium (Current Protocols in Molecular Biology, John Wiley and Sons, Chapter 13.1.2). EBY100 transfected with expression vector pYD2 (Razai A et al. (2005) J Mol Biol 351:158-69) was selected on SD-CAA medium (Current Protocols, Chapter 13). The Aga2p antigen fusion was expressed on the yeast surface by induction in SG-CAA medium (identical to SD-CAA medium except the glucose is replaced by galactose) at 20° C. for 24-48 hr as described previously (Feldhaus M J et al. (2003) Nat Biotechnol 21:163-70). Bacteria strain E. coli DH5α and TG1 were used for the preparation of plasmid DNA and the expression of soluble scFv antibodies respectively. SV5 antibody was purified from hybridoma supernatant using Protein G and directly labeled with ALEXA FLUOR® 488 or ALEXA FLUOR® 647 using a kit provided by the manufacturer (Invitrogen; Carlsbad, Calif.). Biotin conjugated rabbit anti-fd bacteriophage was purchased from Sigma and used to detect phage antibodies. Monoclonal antibody D7 against EphA2 ECD was purchased from Upstate Biotech, polyclonal goat anti-EphA2 and recombinant mouse Ephrin A1 with human Fc fusion protein from R&D Systems, anti-CD44 antibody for Western Blotting from NeoMarkers, and monoclonal anti-CD44 recognizing link domain from Abcam. The full-length cDNA of EphA2 and CD44 was obtained from the ATCC.
The phage library used in the examples below contains non-immune human single-chain Fv antibodies (scFv). Briefly, the library was generated by first constructing a cDNA library from RNAs of human spleen cells and peripheral blood lymphocytes. Heavy chain and light chain repertoires were joined to form the scFV gene repertoire. The single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library was subcloned into a true phage vector to create a multivalently displayed scFv phage library. For details, see Sheets M D et al. (1998) Proc Natl Acad Sci USA 95:6157-62 and O'Connell D et al. (2002) J Mol Biol 321:49-56.
Antigen and Antigen Domains Displayed on the Yeast Surface.
Primers annealing to antigen cDNA and having a 25-mer overlapping sequence with pYD2/NcoI-NotI-digested vector were designed to amplify antigen domains by PCR using Pfu polymerase. After gel purification, the amplified antigen fragment and NcoI-NotI digested pYD2 vector were used to transform LiAc-treated EBY100 cells by gap repair (Gietz R D et al. (1991) Yeast 7:253-63; Orr-Weaver T L et al. (1983) Proc Natl Acad Sci USA 80:4417-21). The transformation mixes were cultured and subcultured in SD-CAA, and induced by culturing in SG-CAA medium for 24-48 hours at 18° C. To validate antigen display, anti-EphA2 (R&D) and recombinant mouse Ephrin A1 (R&D) were analyzed for binding to yeast displayed EphA2 ECD, and anti-CD44 antibody (Abcam) was analyzed for binding to CD44 domain 1 by flow cytometry. Briefly, the induced yeast cells (106 cells) with specific displayed antigen domains were incubated with monoclonal or polyclonal antibodies (1 μg/ml) for 1 h at 4° C., detected using anti-goat PE conjugate for anti-EphA2, anti-human (Fc specific) for rEphrinA1-human Fc fusion protein, and anti-rabbit PE for anti-CD44 respectively, and co-stained with SV5-ALEXA FLUOR® 647 dye.
Optimization of Elution Buffer for Phage Antibody Selection.
Different elution buffer including phosphate buffered saline, pH 7.4 (PBS), 40 mM 2-mercaptoethylamine (2-MEA), 1 mM dithiolthreitol (DTT), 100 mM triethylamine (TEA) and 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5% polysorbate 20/TWEEN® 20 were evaluated for their ability to elute bound phage form the yeast surface. The elution time was 1 hour at 37° C. for PBS, 2-MEA and DTT, and 2 minutes at RT for TEA and glycine. After neutralizing with 10 mM cysteine for 2-MEA and DTT elution, and 1⁄2 volume of 1M Tris-HCl (pH 7.4) for TEA and glycine elutions, the eluted mixture was used to infect exponentially growing E. coli TG1 cells, and the titer of phage determined by serial dilution and plating on tetracycline resistant media.
Selection of Phage Antibodies Specific to Yeast Displayed Antigen Domains.
Human mammary epithelial cell (HMEC), luminal breast cancer cell line SUM52PE, T47D, and MDAMB453 were used to deplete the phage library of nonspecific binders by incubating 1012 phage particles (Sheets M D et al. (1998) Proc Natl Acad Sci USA 95:6157-62; Huie M A et al. (2001) Proc Natl Acad Sci USA 98: 2682-7) with 108 cells for 4 h at 4° C. The depleted phage library was then incubated with 5×106 basal breast cancer cell line MDAMB231 cells for 1 h at 4° C., followed by washing with cold PBS and incubation with 37° C.-prewarmed medium/10% FBS for 30 mM at 37° C. to enable the receptor mediated endocytosis of phage particles. The cell surface was stripped by three incubations of five minutes with 4 ml of glycine buffer (150 mM NaCl, 0.1 M glycine, pH 2.5). The cells were then trypsinized, washed with PBS, lysed with 1 ml of 100 mM TEA for four minutes at 4° C. and neutralized with 0.5 ml of 1M Tris (pH 7.4). Internalized phage (TEA lysate) was amplified for further selections.
After two rounds of selection on MDAMB231 cells, the polyclonal phage antibodies were used to select phage antibodies specific to yeast displayed antigens EphA2 (Y-EphA2) and CD44 link domain (Y-CD44 D1). The induced yeast cells displaying an irrelevant protein were used to deplete the non-specific binders by incubating 2.5×1011 phage particles with 109 yeast cells for 2 h at 4° C. The filtered supernatant containing the depleted phage library was then incubated with 2×107 yeast-cells displaying specific antigen domain for 1 h at 4° C. Yeast cells were washed with cold PBS ten times and pelleted by centrifugation. The bound phage antibodies were eluted by incubating yeast cells with 1 ml of 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5% polysorbate 20/TWEEN® 20, neutralized with 0.5 ml of 1M Tris-HCl (pH 7.4), and amplified for another round of selection. In the second round of selection, 2×107 yeast cells were used for both antigens, while 2.1×1012 phage particles from the first round selection were used for CD44 domain 1 compared to 3.4×1011 used for EphA2. Two rounds of selection were performed.
Characterization of Phage Antibodies.
After two rounds of selection, individual phage antibodies were prepared by growing single colonies in 96-well microtiter plates as described (O'Connell D et al. (2002) J Mol Biol 321:49-56). Binding of each phage antibody to yeast displayed antigen was determined by incubation of 105 yeast cells with 100 μl phage supernatant diluted in FACS buffer (PBS with 1 mM MgCl2, 0.1 mM CaCl2 and 0.3% BSA) for 2 h at 4° C. in conical 96-well microtiter plates, followed by incubation with biotinylated anti-fd antibody and streptavidin-phycoerythrin conjugate (PE) (Jackson), and analyzed using a FACS LSRII (Becton Dickinson). The number of unique phage antibodies was determined by patterns of BstNI digestion of 18 scFv genes amplified by PCR from phage-infected bacteria (Marks, J D et al. (1991) J Mol Biol 222:581-97) and confirmed by DNA sequencing.
For binding to breast cancer cells and Ephrin A1 competition experiments, 5×104 MDAMB231 cells were incubated with 108 phage antibodies in the presence of recombinant mouse Ephrin A1 (R&D) at concentration of 0 to 1000 ng/ml for 2 h at 4° C. The bound phage antibodies were detected by incubating cells with biotin conjugated anti-fd antibody (1 μg/ml) (Sigma) for 30 min at 4° C. and streptavidin-PE (Jackson) followed by flow cytometry analysis.
Immunoprecipitation and Western Blot Using scFv Antibodies.
MDAMB231 cell extracts were prepared using 1 ml of lysis buffer per T75 culture flask, containing 0.5% NP40, 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM DTT, and protease inhibitor cocktail (Sigma). Soluble scFv antibodies with a (His)6 tag at the C-terminal were generated by subcloning scFv genes from the phage vector into the expression vector pUC119mycHis (Schier R et al. (1995) Immunotechnology 1:73-81), followed by purification from the periplasmic fraction of E. coli TG1 by IMAC51 using a Ni-NTA column (Qiagen) and gel filtration (Schier R et al. (1996) J Mol Biol 255:28-43). Cell extracts were incubated with scFv at 26 μg/ml for 2 h at 4° C. before the immune complexes were captured on Ni-NTA agarose. The agarose captured immune complexes were then washed 5 times in lysis buffer and heated to 94° C. for 4 min in non-reducing protein loading buffer. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western Blotting using anti-EphA2 (Upstate) and anti-CD44 (NeoMarkers) antibodies.
Immunofluorescence.
MDAMB231 cells were grown on coverslips to 70% of confluence in 12 well-plates and incubated with 1011 phage antibodies for three hours at 37° C. The coverslips were washed once with PBS, three times for five minutes with glycine buffer (50 mM glycine (pH 2.5), 150 mM NaCl), neutralized with PEM (80 mM Potassium PIPES (pH 6.8), 5 mM EGTA (pH 7), 2 mM MgCl2), and fixed with PEM containing 4% (W/V) paraformaldehyde for 30 min on ice. Cells were quenched with 0.1 M NH4Cl, permeabilized with 0.5% 4-octylphenol polyethoxylate/TRITONTM X-100, and blocked with 5% non-fat dry milk in TBS-T buffer overnight at 4° C. After blocking endogenous biotin with Avidin-Biotin Kit (Lab Vision), intracellular phages were detected with biotinylated anti-fd polyclonal antibody (Sigma) and streptavidin TEXAS RED® (sulforhodamine 101 acid chloride) dye (Amersham). Coverslips were inverted on a slide on mounting medium and microscopic images were taken with a Zeiss LSM 510 laser scanning microscope (Zeiss, Germany).
Example 1
Display of Tumor Associated Antigens on the Surface of Yeast
Two TAA (CD44 and EphA2) are overexpressed in basal breast cancers (Hamilton S R et al. (2007) J Biol Chem 282:16667-80) and were selected for display on the surface of Sachromyces cerevisiae. For yeast surface display, the full-length extracellular domain (ECD) of EphA2 (aa 1-510) and the link domain of CD44 (aa 1-149) (domain 1) were cloned into the yeast display vector pYD2 for (C-terminal) fusion to Aga2 (Razai A et al. (2005) J Mol Biol 351:158-69). Vector DNA was used to transform EBY100, and cell surface display was induced. Both extracellular domains of CD44 and EphA2 were well displayed on the yeast surface as quantitated by a monoclonal antibody to a C-terminal epitope tag (FIG. 1). Specific binding to yeast displayed EphA2 and CD44 extracellular domains of the EphA2 natural ligand Ephrin A1, and antibodies to EphA2 and CD44 suggests that the domains are not only displayed but displayed in a form that can bind to ligand and can be specifically recognized by antibodies (FIG. 1).
Example 2
Efficient Recovery of Antigen Specific Phage Antibodies from Yeast Cell Surface Displayed Antigen
A scFv phage antibody that specifically bound to EphA2 was used to study the ability to select phage antibodies on yeast displayed antigen. Approximately 1011 phage particles displaying anti-EphA2 human scFv 2D6 were incubated with 108 yeast cells displaying the target antigen EphA2 ECD (Y-EphA2). As a control, an identical number of anti-EphA2 phage antibodies were incubated with 108 yeast cells displaying an irrelevant scFv (Y-CON). The recovery of anti-EphA2 phage antibody from yeast displaying the EphA2 ECD was more than 104 fold higher than the recovery of anti-EphA2 phage antibody from yeast displaying the scFv (FIG. 2, panel A). To determine the optimal buffer to elute phage antibodies from the yeast surface, different buffers (PBS, 2-MEA, DTT, triethylamine (TEA) and glycine) were evaluated. Although yeast surface display of TAA results from the disulfide linkage between the Aga2 and Aga1 proteins on the yeast surface, reducing agents, including 2-MEA and DTT, resulted in poorer recovery of viable phage antibodies than spontaneous dissociation of phage by incubation in PBS (FIG. 2, panel B). In contrast, elution with high pH TEA buffer or low pH glycine buffer increased the number of viable phage recovered approximately two fold, with low pH glycine being the optimal elution buffer of those studied. This elution buffer was used for subsequent studies.
To determine the minimum frequency of a specific antibody within a library that can be enriched and selected, phage displayed anti-EphA2 antibodies were serially diluted from 109 to 100 cfu and then mixed with 109 helper phage VCSM13. Phage mixtures were incubated with 107 yeast cells displaying the EphA2 ECD or with yeast displaying the CD44 domain 1, followed by washing, elution and titration of the recovered phages. With an input of 102 specific phage particles, about 12 phage were recovered from yeast cells displaying EphA2, while an input of at least 105 phage were required before phage were present in the output when selected on CD44 (FIG. 2, panel C). The average recovery of phage antibodies was 6.5×10−2 for the specific antigen-antibody pair, and 6.5×10−5 for the mismatched pair (Table 5), respectively. This high recovery ratio for specific compared to non-specific phage suggested that it would be possible to enrich and select phage antibodies on yeast displayed antigen.







TABLE 5







Recovery of specific phage antibody from yeast displayed antigens.


The indicated yeast displayed antigen was incubated with 109


phage and the titer of bound phage determined. Results are


expressed as the ratio of output/input phage.










Ag:Ab
PhAb Output/Input






Y-EphA2 ECD:PhAb-EphA2
6 × 10−2



Y-CD44 D1:PhAb-CD44
7 × 10−2



Y-EphA2 ECD:PhAb-CD44
3 × 10−5



Y-CD44 D1:PhAb-EphA2
1 × 10−6





Y = yeast displayed antigen; PhAb = phage displayed antibody.






Example 3
Selection of Antigen Specific Phage Antibodies on Yeast Cells Displaying Tumor Antigens
The strategy used to select internalizing phage antibodies to specific tumor antigens is shown in FIG. 3. To ensure that phage antibodies bound tumor antigens as presented on the surface of human tumor cells and could be internalized upon antigen binding, a starting phage library was used. The polyclonal phage output after the second round of selection of a non-immune human scFv phage library (O'Connell D et al. (2002) J Mol Biol 321:49-56) was selected for endocytosis into MDAMB231 tumor cells. Based on the fact that basal subtype breast cancer cells over-express EphA2 (Neve R M et al. (2006) Cancer Cell 10:515-27) and CD44 (Hamilton S R et al. (2007) J Biol Chem 282:16667-80), the phage output of selection on the basal subtype breast cancer cell line MDAMB231 was selected independently on yeast displayed EphA2 ECD (aa 25-534) and CD44 domain 1 (aa 21-169). To remove phage antibodies that bound to irrelevant proteins on the yeast surface, 2.5×1011 phage from the second round output of the MDAMB231 selection were incubated first with 1×109 yeast cells displaying the scFv 4E17 (Y-CON). Then, the depleted phage library was incubated with 2×107 yeast cells displaying the relevant antigen (Y—Ag) (FIG. 3, panel A).
For both EphA2 ECD and CD44 domain 1 selections, the number of phage recovered from each yeast cell in the second round of selection increased over 40 fold compared to the first round (Table 6), suggesting enrichment for phage binding yeast displayed tumor antigens. This was verified by using polyclonal phage to stain the yeast displayed antigens. From both the first and second round of selections, polyclonal phage antibodies showed specific binding to the antigen domain that was used for selection, with stronger staining after the second round of selection (FIG. 3, panel B). In contrast, prior to selection, the input phage antibody library gave no signal above background on yeast cells displaying either EphA2 or CD44. Binding was specific for the yeast displayed antigen, since the polyclonal phage did not bind yeast cells displaying an irrelevant protein (the scFv 4E17) (FIG. 3, panel B). To determine the frequency of binding phage antibodies, 96 individual clones were picked, phage produced, and the phage analyzed for binding to the yeast displayed tumor antigen. After one round and two rounds of selection, 31/96 (32.2%) and 39/96 (40.6%) of the clones from EphA2 selection bound yeast cells displaying EphA2 ECD, and 11/96 (11.7%) and 21/96 (21.9%) of the clones from CD44 selection bound yeast displayed CD44, respectively (FIG. 3, panel C)







TABLE 6







Phage display scFv antibody selection on yeast displayed antigens.


Phage input and output ratios during the first and second


rounds of selection on yeast displayed antigens.














Output
Phage


Antigen
Input (cfu)
Output (cfu)
Input
Yeast












Round 1 selection











EphA2-ECD
2.5 × 1011
3.6 × 106
1.3 × 10−5
0.18


CD44-ECD D1
2.5 × 1011
9.8 × 106
3.9 × 10−5
0.49









Round 2 selection











EphA2-ECD
3.4 × 1011
2.4 × 108
 7 × 10−4
12


CD44-ECD D1
2.1 × 1012
4.3 × 108
 2 × 10−4
20









Example 4
Identification and Characterization of Phage Antibodies
Individual phage antibodies from the second round of selection that bound the yeast displayed tumor antigen were analyzed by PCR fingerprinting and DNA sequencing of the scFv genes. The results are shown in FIG. 9, where the nucleic acid and amino acid sequences for each of the isolated clones are shown. The sense strand is shown as the top strand of the nucleic acid sequences in FIG. 9. The CDRs and framework regions are identified based on the Kabat database for each antibody as shown in FIGS. 7 and 8.
Three unique human scFv antibodies (D2-1A7, D2-1A9 and D2-1B1) were identified which bound to EphA2, and two unique scFv (F2-1A6 and F2-1H9) were identified which bound to CD44 ECD domain 1. Characterization of each of these scFv on yeast displayed EphA2-ECD (Y-EphA2 ECD), CD44 ECD domain 1 (Y-CD44 ECD D1) and full-length ECD (Y-CD44 ECD), and scFv 4E17 (Y-CON) indicated that each scFv was specific for its target antigen (FIG. 4, panel A). Each unique phage antibody was also analyzed for its ability to bind the original selecting tumor cell line MDAMB231 by flow cytometry. Each phage antibody strongly stained MDAMB231 cells (FIG. 4, panel B). Since the initial selection of the phage antibody library aimed to target cell surface receptors specific to basal subtype breast cancer cells, the binding of the selected mAbs to basal and luminal breast cancer cell lines was determined. Each mAb was relatively specific for basal breast cancer cell lines compared to luminal breast cancer cell lines (FIG. 4, panel C).
Example 5
Binding Specificity of Phage Antibodies
The binding of the identified EphA2 and CD44 antibodies to native antigens was also confirmed by immunoprecipitation of the receptors from cell extracts of MDAMB231 cells followed by Western blotting with murine monoclonal antibodies specific to EphA2 and CD44 (FIG. 5, panel A). The specificity of the EphA2 mAbs was further studied. The ability of each of the EphA2 mAbs was evaluated for its ability to compete with the natural ligand, ephrin A1 for binding to EphA2 on the surface of MDAMB231 cells. Although the 1050 of ephrin A1 for phage antibodies D2-1A7 and D2-1A9 at the given concentration differed by 9-fold, 1 μg/ml ephrin A1 can fully block the cell binding of both D2-1A7 and D2-1A9 phage antibodies (FIG. 5, panel B), indicating that these two antibodies bind epitopes which overlap with Ephrin A1.
Example 6
Phage Antibodies are Internalized by MDAMB231 Cells
Since the phage antibodies identified by yeast display antigen biopanning were originally selected for the ability to be endocytosed into MDAMB231 cells (FIG. 3, panel A), it was anticipated that they would be internalized efficiently. To determine whether D2-1A7, D2-1A9 and F2-1A6 phage antibodies were endocytosed, the phage antibodies were incubated with MDAMB231 cells at 37° C. to allow receptor mediated endocytosis, the surface bound phage removed by stripping with low pH buffer, and the internalized phage stained with anti-fd antibody and observed using confocal microscopy (FIG. 6). Both D2-1A7 and D2-1A9 anti-EphA2 antibodies gave strong intracellular staining, a control phage gave no staining, and F2-1A6 anti-CD44 antibody gave a different staining pattern than the anti-EphA2 phage.
Example 7
Fd Phage and Phagemid Antibodies Specific for CD44 or EphA2
To select antigen specific antibodies without the use of mammalian cells, the non-immune human scFv phage library (Sheets, 1998; Huie, 2001) was incubated with yeast-displayed tumor associated antigens (TAAs) without prior selection on cancer cells. Specifically, 1012 fd-phage particles (Huie et al. (2001) supra) or 1013 phagemid-phage particles (Sheets et al (1998) supra) were first incubated with 1010 yeast cells displaying an irrelevant protein for 2 h at 4° C. to remove phage antibodies binding common yeast proteins. The depleted phage library was then incubated with 108 yeast cells displaying a specific antigen domain for 2 h at 4° C. (for example EphA2 or CD44). Yeast cells were washed with cold PBS ten times and pelleted by centrifugation. The bound phage antibodies were eluted by incubating yeast cells with 1 ml of 100 mM Glycine/150 mM NaCl/0.1% BSA/0.5% polysorbate 20/TWEEN® 20, neutralized with 0.5 ml of 1M Tris-HCl (pH 7.4), and amplified for another round of selection. In the second round of selection, 2×107 yeast cells were used for both antigens, while phage particles from the first round selection were used as the input phage library with 1011 phage for fd library and 1012 for phagemid library respectively. Two rounds of selection were performed to enrich phage antibodies specific to the TAA used for selection. If two rounds of selection didn't enrich TAA specific antibodies, a third round of selection was performed, following the same protocol as the second round of selection.
The method for screening and characterization of monoclonal phage antibodies was the same as that described in Examples 4 and 5.
Five fd phage antibodies, designated E8H11, E8H7, E8G12, E8F11, and E8C9, were raised to CD44 link domain and were specific for CD44 link domain. Two fd phage antibodies, designated D6G9 and D6D3, were raised to the CD44s (standard form), and were specific for the CD44s.
Two phagemid antibodies, designated D1C5 and D1D1, were raised to the CD44 link domain, and were specific for the CD44 link domain. Six phagemid antibodies, designated HB8, HC2, HC4, HE3, HF1, and HH3, were raised to CD44s and were specific for CD44s.
Five fd phage antibodies, designated A3H9, A3G3, A3D10, A3D1, and A3C8 were raised to EphA2 and were specific for EphA2. Eight phagemid antibodies, designated 1A3, 1A5, 1A8, 1A12, 1B2, 1C2, 1C7, and 1D8, were raised to EphA2 and were specific for EphA2.
Another fd phage antibody, designated 15H11, was raised to EphA2 and was specific to EphA2.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. While the subject antibody, method, and composition have been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A conjugated monoclonal antibody comprising a monoclonal antibody attached to a polymer comprising poly(ethylene glycol) (PEG), wherein the monoclonal antibody specifically binds an epitope of ephrinA receptor A2 (EphA2) that is specifically bound by a monoclonal antibody comprising:
a variable heavy chain (VH) polypeptide comprising:
a VH CDR1 comprising the amino acid sequence SYAMH (SEQ ID NO:29),
a VH CDR2 comprising the amino acid sequence VISYDGSNKYYADSVKG (SEQ ID NO: 45), and
a VH CDR3 comprising the amino acid sequence ASVGATGPFDI (SEQ ID NO: 42); and


a variable light chain (VL) polypeptide comprising:
a VL CDR1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO:17),
a VL CDR2 comprising the amino acid sequence GENNRPS (SEQ ID NO:71), and

a VL CDR3 comprising the amino acid sequence NSRDSSGTHLTV (SEQ ID NO: 60).

2. The conjugated monoclonal antibody of claim 1, wherein the monoclonal antibody is covalently bound to the polymer.

3. The conjugated monoclonal antibody of claim 1, wherein the monoclonal antibody is covalently bound to a terminus of the polymer.

4. The conjugated monoclonal antibody of claim 1, wherein the monoclonal antibody comprises an Fc region and the Fc region of the monoclonal antibody is attached to the polymer.

5. The conjugated monoclonal antibody of claim 1, wherein the variable heavy chain (VH) polypeptide and the variable light chain (VL) polypeptide of the monoclonal antibody are joined in a single chain Fv (scFv).

6. The conjugated monoclonal antibody of claim 1, wherein the polymer is soluble in water at room temperature.

7. The conjugated monoclonal antibody of claim 5, wherein the conjugated scFv is characterized by one or more of the following:
the scFv is covalently bound to a terminus of the polymer;
the scFv is covalently bound to a terminus of the polymer via a linker; and
the polymer is soluble in water at room temperature.

8. The conjugated monoclonal antibody of claim 1, wherein the conjugated monoclonal antibody is a human, humanized, or chimeric monoclonal antibody.

9. A composition comprising a pharmaceutically acceptable carrier and a conjugated monoclonal antibody of claim 1.

10. The composition of claim 9, wherein said composition is formulated for parenteral administration.

11. The conjugated monoclonal antibody of claim 5, wherein the variable light chain (VL) polypeptide and the variable heavy chain (VH) polypeptide of the scFv are human or humanized.

12. The conjugated monoclonal antibody of claim 7, wherein the variable light chain (VL) polypeptide and the variable heavy chain (VH) polypeptide of the scFv are human or humanized.

13. A composition comprising a pharmaceutically acceptable carrier and a conjugated monoclonal antibody of claim 11.

14. The composition of claim 13, wherein said composition is formulated for parenteral administration.

15. A composition comprising a pharmaceutically acceptable carrier and a conjugated monoclonal antibody of claim 12.

16. The composition of claim 15, wherein said composition is formulated for parenteral administration.

17. The conjugated monoclonal antibody of claim 1, wherein the monoclonal antibody is an IgG, Fab, F(ab′)2, or (scFv′)2.