Patent Application
20220213214
This application contains a Sequence Listing submitted as an electronic text file named “18-285-PCT_Sequence-Listing_ST25.txt”, having a size in bytes of 30 kb, and created on May 13, 2020. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).
Innervated tumors are more aggressive than less innervated one. For instance, in prostate cancer, recruitment of nerve fibers to cancer tissue is associated with higher tumor proliferative indices and a higher risk of recurrence and metastasis. Denervation studies in pre-clinical and genetically engineered mouse cancer models support a functional contribution of neural elements in disease progression. These studies strongly indicate that the nervous system is not a bystander but instead an active participant in carcinogenesis and cancer progression.
In one aspect, the disclosure provides isolated anti-human ephrin B1 antibody, or fragment thereof, comprising a heavy chain that comprises 1, 2, or all 3 complementarity determining regions (CDR) selected from the group consisting of:
Heavy chain CDR1 (H-CDR1) comprising the amino acid sequence selected from the group consisting of (T/D)(Y/F)(N/Y)(V/I/M)(H/N) (SEQ ID NO:1), TYN(V/I)H (SEQ ID NO:2), TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
Heavy chain CDR2 (H-CDR2) comprising the amino acid sequence selected from the group consisting of (F/-)(I/-)(R/-)(A/N)(M/K)(W/V)NG(G/Y)(R/G/T)TDYN(S/P)(A/S)(F/V)K(S/G) (SEQ ID NO:6), AMWNGG(R/G)TDYNSAFKS (SEQ ID NO:7), AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
Heavy chain CDR3 (H-CDR3) comprising the amino acid sequence selected from the group consisting of (E/-)(D/-)(Y/-)(Y/-)(Y/-)(S,-)(G/-)(R/-)(L/P/F)(D/I)(D/Y) (SEQ ID NO:11), LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12).
In various embodiments, the heavy chain comprises 1, 2, or all 3 of the CDRS selected from the group consisting of:
H-CDR1 comprises the amino acid sequence selected from the group consisting of TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence selected from the group consisting of AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
H-CDR3 comprises the amino acid sequence selected from the group consisting of LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12); or
H-CDR1 comprises the amino acid sequence of TYNVH (SEQ ID NO:3);
H-CDR2 comprises the amino acid sequence of AMWNGGRTDYNSAFKS (SEQ ID NO:8); and
H-CDR3 comprises the amino acid sequence of LDY; or
H-CDR1 comprises the amino acid sequence of TYNIH (SEQ ID NO:4);
H-CDR2 comprises the amino acid sequence of AMWNGGGTDYNSAFKS (SEQ ID NO:9); and
H-CDR3 comprises the amino acid sequence of PTD; or
H-CDR1 comprises the amino acid sequence of DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence of FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
H-CDR3 comprises the amino acid sequence of EDYYYSGRFDY (SEQ ID NO:12).
In another embodiment, the isolated anti-human ephrin B1 antibody, or fragment thereof, comprises a light chain that comprises 1, 2, or all 3 complementarity determining regions (CDR) selected from the group consisting of:
Light chain CDR1 (L-CDR1) comprises the amino acid sequence selected from the group consisting of KASQ(S/N)(V/I)(G/N)(I/K)(D/N/Y)(V/L)D (SEQ ID NO:13), KASQSVGI(D/N)VD (SEQ ID NO:14), KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
Light chain CDR2 (L-CDR2) comprises the amino acid sequence selected from the group consisting of (G/H)(A/T)(S/N)(S/N)(R/L)(H/T)T (SEQ ID NO:18), GAS(N/S)RHT (SEQ ID NO:19), GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
Light chain CDR3 (L-CDR3): selected from the group consisting of L(H/Q)(Y/H)(G/D)S(I/R)P(F/R)T (SEQ ID NO:23), LHYGSIPFT (SEQ ID NO:24), and LQHDSRPRT (SEQ ID NO:25).
In various embodiments, the light chain comprises 1, 2, or all 3 of the CDRS selected from the group consisting of:
L-CDR1 comprises the amino acid sequence selected from the group consisting of KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence selected from the group consisting of GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence selected from the group consisting of LHYGSIPFT(SEQ ID NO:24) and LQHDSRPRT (SEQ ID NO:25); or
L-CDR1 comprises the amino acid sequence of KASQSVGIDVD (SEQ ID NO:15);
L-CDR2 comprises the amino acid sequence of GASNRHT (SEQ ID NO:20); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24); or
L-CDR1 comprises the amino acid sequence of KASQSVGINVD (SEQ ID NO:16);
L-CDR2 comprises the amino acid sequence of GASSRHT (SEQ ID NO:21); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24); or
L-CDR1 comprises the amino acid sequence of KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence of HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence of LQHDSRPRT (SEQ ID NO:25).
In another embodiment, the isolated anti-human ephrin B1 antibody, or fragment thereof, comprising at least 1, 2, 3, 4, 5, or all 6 complementarity determining regions (CDR) selected from the group consisting of:
Heavy chain CDR1 (H-CDR1) comprises the amino acid sequence selected from the group consisting of (T/D)(Y/F)(N/Y)(V/I/M)(H/N) (SEQ ID NO:1), TYN(V/I)H (SEQ ID NO:2), TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
Heavy chain CDR2 (H-CDR2) comprises the amino acid sequence selected from the group consisting of (F/-)(I/-)(R/-)(A/N)(M/K)(W/V)NG(G/Y)(R/G/T)TDYN(S/P)(A/S)(F/V)K(S/G)(SEQ ID NO:6), AMWNGG(R/G)TDYNSAFKS (SEQ ID NO:7), AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
Heavy chain CDR3 (H-CDR3) comprises the amino acid sequence selected from the group consisting of (E/-)(D/-)(Y/-)(Y/-)(Y/-)(S/-)(G/-)(R/-)(L/P/F)(D/T)(D/Y) (SEQ ID NO:11), LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12);
Light chain CDR1 (L-CDR1) comprises the amino acid sequence selected from the group consisting of KASQ(S/N)(V/I)(G/N)(I/K)(D/N/Y)(V/L)D (SEQ ID NO:13), KASQSVGI(D/N)VD (SEQ ID NO:14), KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
Light chain CDR2 (L-CDR2) comprises the amino acid sequence selected from the group consisting of (G/H)(A/T)(S/N)(S/N)(R/L)(H/T)T (SEQ ID NO:18), GAS(N/S)RHT (SEQ ID NO:19), GASNRHT (SEQ ID NO:20). GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
Light chain CDR3 (L-CDR3): selected from the group consisting of L(H/Q)(Y/H)(G/D)S(I/R)P(F/R)T (SEQ ID NO:23), LHYGSIPFT (SEQ ID NO:24), and LQHDSRPRT (SEQ ID NO:25).
In various embodiments, at least 1, 2, 3, 4, 5, or all 6 of the CDRS are selected from the group consisting of:
H-CDR1 comprises the amino acid sequence selected from the group consisting of TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence selected from the group consisting of AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
H-CDR3 comprises the amino acid sequence selected from the group consisting of LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12);
L-CDR1 comprises the amino acid sequence selected from the group consisting of KASQSVGIDVD (SEQ ID NO: 15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence selected from the group consisting of GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence selected from the group consisting of LHYGSIPFT(SEQ ID NO:24) and LQHDSRPRT (SEQ ID NO:25); or
H-CDR1 comprises the amino acid sequence of TYNVH (SEQ ID NO:3);
H-CDR2 comprises the amino acid sequence of AMWNGGRTDYNSAFKS (SEQ ID NO:8);
H-CDR3 comprises the amino acid sequence of LDY;
L-CDR1 comprises the amino acid sequence of KASQSVGIDVD (SEQ ID NO:15);
L-CDR2 comprises the amino acid sequence of GASNRHT (SEQ ID NO:20); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24); or
H-CDR1 comprises the amino acid sequence of TYNIH (SEQ ID NO:4);
H-CDR2 comprises the amino acid sequence of AMWNGGGTDYNSAFKS (SEQ ID NO:9);
H-CDR3 comprises the amino acid sequence of PTD;
L-CDR1 comprises the amino acid sequence of KASQSVGINVD (SEQ ID NO:16);
L-CDR2 comprises the amino acid sequence of GASSRHT (SEQ ID NO:21); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24); or
H-CDR1 comprises the amino acid sequence of DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence of FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
H-CDR3 comprises the amino acid sequence of EDYYYSGRFDY (SEQ ID NO:12);
L-CDR1 comprises the amino acid sequence of KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence of HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence of LQHDSRPRT (SEQ ID NO:25).
In other embodiments, the anti-human ephrin B1 antibody or fragment thereof comprises a heavy chain having the amino acid sequence selected from the group consisting of the following, wherein residues in parentheses are optional:
GFSLT
TYNVHWVRQTPGKGLEWMGAMWNGGRTDYNSAFKSRLSISR
DTSKSQVFLNMNSLQADDTAKYFCAR
LDYWGQGVMVTVAS
GFSLT
TYNIHWVRQPPGKGLEWMGAMWNGGGTDYNSAFKSRLSISR
DTSKSQVFLKMNSLQTDDTAKYFCAT
PTDWGQGVMVTVSS
GFTFT
DFYMNWIRQPAGKAPEWLGFIRNKVNGYTTDYMPSVKGRFT
ISRENTQNMLYLQMNTLRAEDTATYYCTR
EDYYYSGRFDYWGQGVM
VTVSS
In other embodiments, the anti-human ephrin B1 antibody or fragment thereof comprises a light chain having the amino acid sequence selected from the group consisting of the following, wherein residues in parentheses are optional:
DLAVYYC
LHYGSIPFTFGSGTKLEIK
EDLAVYYC
LHYGSIPFTFGSGTRLEIK
DVATYFC
LQHDSRPRTFGGGTKLDLK
In various further embodiments, the disclosure provides anti-human ephrin B1 antibody or fragment thereof, wherein the antibody or fragment thereof binds selectively to a human ephrin B1 epitope within the amino acid sequence consisting of SEQ ID NO: 32. (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL).
In various other embodiments, the antibody comprises a monoclonal antibody, or fragment thereof; the antibody comprises a humanized antibody, or fragment thereof; and/or the antibody or fragment thereof further comprises a detectable label and/or a therapeutic agent conjugated to the antibody or fragment thereof.
In other embodiments, the disclosure provides nucleic acids encoding an antibody of the disclosure, an expression vector comprising the nucleic acid of the disclosure operatively linked to a suitable control sequence, host cells comprising the expression vector or the nucleic acid of the disclosure, pharmaceutical compositions comprising an antibody or fragment thereof of the disclosure and a pharmaceutically acceptable carrier, and methods for treating tumors comprising administering to a subject having a tumor with an amount effective to limit tumor growth or metastasis of the anti-human ephrin B1 antibody or fragment thereof, or the pharmaceutical composition, or any embodiment or combination of embodiments disclosed herein.
As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.
As used herein, “about” means+/−5% of the recited dimension or unit.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
In a first aspect, the disclosure provides isolated anti-human ephrin B1 antibodies, or ephrin B1-binding fragments thereof (referred to herein as “fragments thereof”). The anti-human ephrin B1 antibodies or fragments thereof are useful, for example, to limit tumor growth or metastasis.
In one embodiment, the ephrin B1-specific antibodies bind to an ephrin B1 protein having the sequence shown SEQ ID NO:36 below.
Extracellular domain bold/underlined; cytoplasmic domain italicized; transmembrane domain in enlarged/outline font
In another embodiment, the ephrin B1-specific antibodies bind to one or more epitopes in the extracellular domain (ECD) of an ephrin B1 protein, where the ECD sequence comprises or consists of the sequence shown as SEQ ID NO:37 below:
In another embodiment the ephrin B1-specific antibodies bind to an ephrin B1 protein having the amino acid sequence of SEQ ID NO:36 but which have one or more of the following amino acid changes relative to the amino acid sequence of SEQ ID NO:36:
These positional changes are present in variants of the human EphrinB1 protein (SEQ ID NO:36), such as variants associated with craniofrontonasal syndrome.
As disclosed herein, “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an epitope in human EphrinB1 protein. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. Such antibody or antibody fragments thereof may include, but are not limited to monoclonal antibodies, humanized antibodies, chimeric antibodies, Fab′, F(ab′)2, Fab, Fv, rIgG, recombinant single chain Fv fragments (scFv), bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.
As used herein, “isolated” means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term “isolated” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
In one embodiment, the anti-human ephrin B1 antibody, or fragment thereof, comprises a heavy chain that comprises 1, 2, or all 3 complementarity determining regions (CDR) selected from the group consisting of:
Heavy chain CDR1 (H-CDR1) comprising the amino acid sequence selected from the group consisting of (T/D)(Y/F)(N/Y)(V/I/M)(H/N) (SEQ ID NO:1), TYN(V/I)H (SEQ ID NO:2), TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
Heavy chain CDR2 (H-CDR2) comprising the amino acid sequence selected from the group consisting of (F/-)(I-)(R/-)(A/N)(M/K)(W/V)NG(G/Y)(R/G/T)TDYN(S/P)(A/S)(F/V)K(S/G) (SEQ ID NO:6), AMWNGG(R/G)TDYNSAFKS (SEQ ID NO:7), AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
Heavy chain CDR3 (H-CDR3) comprising the amino acid sequence selected from the group consisting of (E/-)(D/-)(Y/-)(Y/-)(Y/-)(S/-)(G/-)(R/-)(L/P/F)(D/T)(D/Y) (SEQ ID NO:11), LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12).
In another embodiment, the heavy chain comprises 1, 2, or all 3 of the CDRS selected from the group consisting of:
H-CDR1 comprises the amino acid sequence selected from the group consisting of TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence selected from the group consisting of AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
H-CDR3 comprises the amino acid sequence selected from the group consisting of LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12).
In a further embodiment, the heavy chain comprises 1, 2, or all 3 of the CDRS as follows:
H-CDR1 comprises the amino acid sequence of TYNVH (SEQ ID NO:3);
H-CDR2 comprises the amino acid sequence of AMWNGGRTDYNSAFKS (SEQ ID NO:8); and
H-CDR3 comprises the amino acid sequence of LDY.
In one embodiment, the heavy chain comprises 1, 2, or all 3 of the CDRS as follows:
H-CDR1 comprises the amino acid sequence of TYNIH (SEQ ID NO:4);
H-CDR2 comprises the amino acid sequence of AMWNGGGTDYNSAFKS (SEQ ID NO:9); and
H-CDR3 comprises the amino acid sequence of PTD.
In another embodiment, the heavy chain comprises 1, 2, or all 3 of the CDRS as follows:
H-CDR1 comprises the amino acid sequence of DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence of FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10); and
H-CDR3 comprises the amino acid sequence of EDYYYSGRFDY (SEQ ID NO:12).
In another embodiment, the disclosure provides isolated anti-human ephrin B1 antibodies, or fragment thereof, comprising a light chain that comprises 1, 2, or all 3 complementarity determining regions (CDR) selected from the group consisting of:
Light chain CDR1 (L-CDR1) comprises the amino acid sequence selected from the group consisting of KASQ(S/N)(V/I)(G/N)(I/K)(D/N/Y)(V/L)D (SEQ ID NO:13), KASQSVGI(D/N)VD (SEQ ID NO:14), KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
Light chain CDR2 (L-CDR2) comprises the amino acid sequence selected from the group consisting of (G/H)(A/T)(S/N)(S/N)(R/L)(H/T)T (SEQ ID NO:18), GAS(N/S)RHT (SEQ ID NO:19), GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
Light chain CDR3 (L-CDR3): selected from the group consisting of L(H/Q)(Y/H)(G/D)S(I/R)P(F/R)T (SEQ ID NO:23), LHYGSIPFT (SEQ ID NO:24), and LQHDSRPRT (SEQ ID NO:25).
In one embodiment, the light chain comprises 1, 2, or all 3 of the CDRS selected from the group consisting of:
L-CDR1 comprises the amino acid sequence selected from the group consisting of KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence selected from the group consisting of GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence selected from the group consisting of LHYGSIPFT(SEQ ID NO:24) and LQHDSRPRT (SEQ ID NO:25).
In another embodiment, the light chain comprises 1, 2, or all 3 of the CDRS are as follows:
L-CDR1 comprises the amino acid sequence of KASQSVGIDVD (SEQ ID NO:15);
L-CDR2 comprises the amino acid sequence of GASNRHT (SEQ ID NO:20); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24).
In a further embodiment, the light chain comprises 1, 2, or all 3 of the CDRS are as follows:
L-CDR1 comprises the amino acid sequence of KASQSVGINVD (SEQ ID NO:16);
L-CDR2 comprises the amino acid sequence of GASSRHT (SEQ ID NO:21); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24).
In one embodiment, the light chain comprises 1, 2, or all 3 of the CDRS are as follows:
L-CDR1 comprises the amino acid sequence of KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence of HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence of LQHDSRPRT (SEQ ID NO:25).
In another embodiment, the isolated anti-human ephrin B1 antibodies, or fragments thereof, comprise at least 1, 2, 3, 4, 5, or all 6 complementarity determining regions (CDR) selected from the group consisting of:
Heavy chain CDR1 (H-CDR1) comprising the amino acid sequence selected from the group consisting of (T/D)(Y/F)(N/Y)(V/I/M)(H/N) (SEQ ID NO:1). TYN(V/I)H (SEQ ID NO:2), TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
Heavy chain CDR2 (H-CDR2) comprising the amino acid sequence selected from the group consisting of (F/-)(I/-)(R/-)(A/N)(M/K)(W/V)NG(G/Y)(R/G/T)TDYN(S/P)(A/S)(F/V)K(S/G) (SEQ ID NO:6), AMWNGG(R/G)TDYNSAFKS (SEQ ID NO:7), AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
Heavy chain CDR3 (H-CDR3) comprising the amino acid sequence selected from the group consisting of (E/-)(D/-)(Y/-)(Y/-)(Y/-)(S/-)(G/-)(R/-)(L/P/F)(D/T)(D/Y) (SEQ ID NO:11), LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12);
Light chain CDR1 (L-CDR1) comprising the amino acid sequence selected from the group consisting of KASQ(S/N)(V/I)(G/N)(I/K)(D/N/Y)(V/L)D (SEQ ID NO:13), KASQSVGI(D/N)VD (SEQ ID NO:14), KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
Light chain CDR2 (L-CDR2) comprising the amino acid sequence selected from the group consisting of (G/H)(A/T)(S/N)(S/N)(R/L)(H/T)T (SEQ ID NO:18), GAS(N/S)RHT (SEQ ID NO:19), GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
Light chain CDR3 (L-CDR3): comprising the amino acid sequence selected from the group consisting of L(H/Q)(Y/H)(G/D)S(I/R)P(F/R)T (SEQ ID NO:23), LHYGSIPFT (SEQ ID NO:24), and LQHDSRPRT (SEQ ID NO:25).
In a further embodiment, at least 1, 2, 3, 4, 5, or all 6 of the CDRS are selected from the group consisting of:
H-CDR1 comprising the amino acid sequence selected from the group consisting of TYNVH (SEQ ID NO:3), TYNIH (SEQ ID NO:4), and DFYMN (SEQ ID NO:5);
H-CDR2 comprising the amino acid sequence selected from the group consisting of AMWNGGRTDYNSAFKS (SEQ ID NO:8), AMWNGGGTDYNSAFKS (SEQ ID NO:9), and FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
H-CDR3 comprising the amino acid sequence selected from the group consisting of LDY, PTD, and EDYYYSGRFDY (SEQ ID NO:12);
L-CDR1 comprising the amino acid sequence selected from the group consisting of KASQSVGIDVD (SEQ ID NO:15), KASQSVGINVD (SEQ ID NO:16), and KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprising the amino acid sequence selected from the group consisting of GASNRHT (SEQ ID NO:20), GASSRHT (SEQ ID NO:21), and HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprising the amino acid sequence selected from the group consisting of LHYGSIPFT(SEQ ID NO:24) and LQHDSRPRT (SEQ ID NO:25).
In another embodiment, at least 1, 2, 3, 4, 5, or all 6 of the CDRS are as follows:
H-CDR1 comprises the amino acid sequence of TYNVH (SEQ ID NO:3);
H-CDR2 comprises the amino acid sequence of AMWNGGRTDYNSAFKS (SEQ ID NO:8);
H-CDR3 comprises the amino acid sequence of LDY;
L-CDR1 comprises the amino acid sequence of KASQSVGIDVD (SEQ ID NO:15);
L-CDR2 comprises the amino acid sequence of GASNRHT (SEQ ID NO:20); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24).
In one embodiment, at least 1, 2, 3, 4, 5, or all 6 of the CDRS are as follows:
H-CDR1 comprises the amino acid sequence of TYNIH (SEQ ID NO:4);
H-CDR2 comprises the amino acid sequence of AMWNGGGTDYNSAFKS (SEQ ID NO:9);
H-CDR3 comprises the amino acid sequence of PTD;
L-CDR1 comprises the amino acid sequence of KASQSVGINVD (SEQ ID NO:16);
L-CDR2 comprises the amino acid sequence of GASSRHT (SEQ ID NO:21); and
L-CDR3 comprises the amino acid sequence of LHYGSIPFT (SEQ ID NO:24).
In a further embodiment, at least 1, 2, 3, 4, 5, or all 6 of the CDRS are as follows:
H-CDR1 comprises the amino acid sequence of DFYMN (SEQ ID NO:5);
H-CDR2 comprises the amino acid sequence of FIRNKVNGYTTDYNPSVKG (SEQ ID NO:10);
H-CDR3 comprises the amino acid sequence of EDYYYSGRFDY (SEQ ID NO:12);
L-CDR1 comprises the amino acid sequence of KASQNINKYLD (SEQ ID NO:17);
L-CDR2 comprises the amino acid sequence of HTNNLQT (SEQ ID NO:22); and
L-CDR3 comprises the amino acid sequence of LQHDSRPRT (SEQ ID NO:25).
In one embodiment, the anti-human ephrin B1 antibody or fragment thereof comprises a heavy chain having the amino acid sequence selected from the group consisting of the following, wherein residues in parentheses are optional:
WMG
AMWNGGRTDYNSAFKSRLSISRDTSKSQVFLNMNSLQADDTAKYFCARLDYWGQGVMVTVAS
LEWMG
AMWNGGGTDYNSAFKSRLSISRDTSKSQVFLKMNSLQTDDTAKYFCATPTDWGQGVMVTV
SS
PEWLG
FIRNKVNGYTTDYMPSVKGRFTISRENTQNMLYLQMNTLRAEDTATYYCTREDYYYSGRF
In another embodiment, the anti-human ephrin B1 antibody or fragment thereof comprises a light chain having the amino acid sequence selected from the group consisting of the following, wherein residues in parentheses are optional:
LLIY
GASNRHTGVPDRFTGSGFGRDFTLTISNVEAEDLAVYYCLHYGSIPFTFGSGTKLEIK
PKLLIY
GASSRHTGVPDRFTGSGFGRDFTLTITNVEAEDLAVYYCLHYGSIPFTFGSGTRLEIK
KLLIY
HTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCLQHDSRPRTFGGGTKLDLK
In a further embodiment, the anti-human ephrin B1 antibody or fragment thereof comprises a heavy chain having the amino acid sequence below, wherein residues in parentheses are optional:
WMG
AMWNGGRTDYNSAFKSRLSISRDTSKSQVFLNMNSLQADDTAKYFCARLDYWGQGVMVTVAS;
LLIY
GASNRHTGVPDRFTGSGFGRDFTLTISNVEAEDLAVYYCLHYGSIPFTFGSGTKLEIK.
In one embodiment, the anti-human ephrin B1 antibody or fragment thereof comprises a heavy chain having the amino acid sequence below, wherein residues in parentheses are optional:
MNSLQTDDTAKYFCAT
PTDWGQGVMVTVSS;
a light chain having the amino acid sequence below, wherein residues in parentheses are optional:
PKLLIY
GASSRHTGVPDRFTGSGEGRDFTLTITNVEAEDLAVYYCLHYGSIPFTFGSGTRLEIK.
In another embodiment, the anti-human ephrin B1 antibody or fragment thereof comprises a heavy chain having the amino acid sequence below, wherein residues in parentheses are optional:
PEWLG
FIRNKVNGYTTDYMPSVKGRFTISRENTQNMLYLQMNTLRAEDTATYYCTREDYYYSGRF
a light chain having the amino acid sequence below, wherein residues in parentheses are optional:
KLLIY
HTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCLQHDSRPRTFGGGTKLDKK.
In one embodiment of all of the above, optional amino acid residues are absent. In another embodiment, optional amino acid residues are present.
In another embodiment, the anti-human ephrin B1 antibody or fragment thereof binds to a human ephrin B1 epitope within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In one embodiment, the antibody or fragment thereof binds to a human ephrin B1 epitope of at least 14 amino acids in length within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In another embodiment, the antibody or fragment thereof binds to a human ephrin B1 epitope of between 14 amino acids and 20 amino acids in length within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In a further embodiment, the human ephrin B1 epitope consists of the amino acid sequence selected from the group consisting of SEQ ID NOS:33-35 SWSSLNPKFLSGKG (SEQ ID NO:33); KNLEPVSWSSLNPKFLSGKG (SEQ ID NO:34); and SGKGLVIYPKIGDKL (SEQ ID NO:35).
In another aspect, the disclosure provides antibodies and fragments thereof that selectively bind to the same epitope as any of the anti-human ephrin B1 antibodies or antigen-binding fragments thereof of the present disclosure. In one embodiment, the anti-human ephrin B1 antibodies or fragments bind selectively to a human ephrin B1 epitope within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In one embodiment, the human ephrin B1 epitope is at least 14 amino acids in length within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In another embodiment, the human ephrin B1 epitope is between 14 amino acids and 20 amino acids in length within the amino acid sequence consisting of SEQ ID NO: 32 (KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL). In a further embodiment, the human ephrin B1 epitope consists of the amino acid sequence selected from the group consisting of SEQ ID NOS:33-35.
The anti-human ephrin B1 antibodies or fragments thereof may be linked with any additional component as deemed suitable for an intended use. In one embodiment, the anti-human ephrin B1 antibody or fragment thereof further comprises a detectable label. Such embodiments may be useful for monitoring a course of treatment, for example. Any suitable detectable label may be bound, either covalently, genetically, or by any other means, to the antibody, including but not limited to radioactive isotopes, fluorescent molecules, magnetic particles (including nanoparticles), metal particles (including nanoparticles), phosphorescent molecules, and enzymes.
In other embodiments, the anti-human ephrin B1 antibody or fragment thereof may further comprise a therapeutic agent conjugated to the antibody or fragment thereof. Any suitable additional therapeutic agent for a given purpose may be linked, including but not limited to an inhibitor of tumor exosomal release (including but not limited to an inhibitor of Rab27a, an inhibitor of Rab27b, and/or GW4869, or pharmaceutical salts thereof); an inhibitor of the interaction between E6 and PTPN13 (including but not limited to peptides that compete with E6 for binding to PTPN13, or that compete with PTPN13 for binding to E6); and/or inhibitors of ephrin B1 phosphorylation (including but not limited to Src kinase inhibitors, including but not limited to dasatanib or a pharmaceutically acceptable salt thereof). The structure of GW4869 is provided below, and its use for exosomal release is provided in Chen et al., Journal of Cell Commun Signal 2018 12: 343-357 PMID 29063370; Richards et al. Oncogene 2017 36: 1770-1778, PMID 27669441; Essandoh et al., Biochim Biophys Acta 2015 1852: 2362-71 PMID 26300484; and Gong et al., Oncotarget 2017 8: 45200-45212 PMID 28423355.
In another aspect isolated nucleic acids are disclosed encoding the antibody of any embodiment or combination of embodiments disclosed herein. The isolated nucleic acid sequence may comprise RNA or DNA. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals.
In a further aspect recombinant expression vectors comprising the isolated nucleic acid of the disclosure are provided. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product. The promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector is replicable in a suitable host organism either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector comprises a plasmid or viral vector.
In a further aspect recombinant host cells comprising the nucleic acid vector of the disclosure are provided. The host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. In one embodiment, the cells are hybridoma cells that express and secrete antibodies of the present disclosure. Thus, the recombinant host cells can be used, for example in methods for producing antibody, comprising:
(a) culturing the recombinant host cell under conditions suitable for expression of the nucleic-acid encoded antibody; and
(b) isolating the antibody from the cultured cells.
Suitable conditions for expression of the nucleic-acid encoded antibody can be determined by those of skill in the art based on the teachings herein, the specific host cells and vectors used, and the general knowledge of those of skill in the art.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes disclosed herein. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes disclosed herein.
In one aspect pharmaceutical compositions are provided, comprising:
(a) the antibody, isolated nucleic acid, recombinant expression vector, or host cell of any embodiment or combination of embodiments disclosed herein; and
(b) a pharmaceutically acceptable carrier.
For example, the antibodies of the disclosure may be present in a pharmaceutical formulation. In this embodiment, the antibodies are combined with a pharmaceutically acceptable carrier. Suitable acids which are capable of forming such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).
The pharmaceutical composition may comprise in addition to the composition of the invention (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
The pharmaceutical compositions of the invention may be made up in any suitable formulation, preferably in formulations suitable for administration by injection. Such pharmaceutical compositions can be used, for example, in the therapeutic methods disclosed herein.
The pharmaceutical compositions may contain any other components as deemed appropriate for a given use, such as additional therapeutics. In one embodiment, the pharmaceutical compositions further comprise an inhibitor of tumor exosomal release, or a pharmaceutically acceptable salt thereof. In one embodiment, the inhibitor of tumor exosomal release comprises an inhibitor of Rab27a and/or an inhibitor of Rab27b. In another embodiment,
the inhibitor of Rab27a and/or the inhibitor of Rab27b are selected from the group consisting Rab27a and/or Rab27b-specific antibodies, aptamers, small interfering RNAs, small internally segmented interfering RNAs, short hairpin RNAs, microRNAs, and/or antisense oligonucleotides.
In another aspect are provided methods for tumor treatment comprising administering to a subject having a tumor an amount effective to limit tumor growth or metastasis of the anti-human ephrin B1 antibody or fragment thereof, or the pharmaceutical composition, of any embodiment or combination of embodiments disclosed herein. As shown in the examples that follow, the inventors have demonstrated that the antibodies of the disclosure are useful to limit tumor growth or metastasis.
As used here, the terms “treating tumor growth” means (i) limiting tumor size, (ii) limiting the rate of increase in tumor size, (iii) reducing tumor innervation, (iv) limiting the rate of increase in tumor innervation, (v) limiting tumor metastases, (vi) limiting the rate of increase in tumor metastases, (vii) limiting side effects caused by tumors (i.e., pain, sickness behavior, etc.), and/or (viii) limiting the rate of increase of side effects caused by tumors.
The amount effective of the anti-human ephrin B1 antibody or fragment thereof to be administered is any amount that will achieve the goal of treating the tumor, and can be determined by one of skill in the art (such as an attending physician) in light of all circumstances, including but not limited to the type of tumor, the subject's condition, other therapeutic treatments that the subject is undergoing (i.e.: chemotherapy, radiation therapy, surgery to remove the tumor, etc.), and all other contributing factors.
As used herein, the term “subject” or “patient” is meant any subject for which therapy is desired, including humans, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, chickens, and so on. Most preferably, the subject is human.
In one embodiment, the methods serve to limit tumor innervation. As used herein, “tumor innervation” is defined as neural fibers invading in, around and/or through a tumor mass. As used herein, “limiting innervation” is defined to include any reduction (i.e., 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or greater reduction) in neo-innervation or existing innervation, compared to no treatment with the anti-human ephrin B1 antibody or fragment thereof.
In one embodiment, the methods of the disclosure further comprise administering to the subject an amount effective of an inhibitor of tumor exosomal release. Any suitable inhibitor of tumor exosomal release may be used, including but not limited to inhibitors of Rab27a and/or Rab27b. Rab27a and Rab27b are members of the small GTPase Rab family that functions in the release of exosomes. In this embodiment, the inhibitor of Rab27a and/or the inhibitor of Rab27b include, but are not limited to Rab27a and/or the Rab27b-specific antibodies, aptamers, small interfering RNAs, small internally segmented interfering RNAs, short hairpin RNAs, microRNAs, antisense oligonucleotides, and/or small molecule inhibitors. The amino acid sequence of human Rab27a and Rab27b are provided below.
The methods of the disclosure may be used to treat any suitable tumor type. In one embodiment, the tumor may be any innervated solid tumor. In various non-limiting embodiments, the methods may be used to treat head, neck, breast, lung, liver, ovaries, colon, colorectal, melanoma, brain or prostate tumors. In a further embodiment, the tumor may be a human papillomavirus (HPV)-positive tumor, including but not limited to HPV+ tumors of the head or neck, and/or cervical cancer. In a further non-limiting embodiment, the HPV+ tumor of the head or neck comprises a squamous cell carcinoma. In another embodiment, the tumor is positive for a high risk HPV, such as HPV16, 18, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68. High risk HPV subtypes all have E6 proteins that contain a C-terminal PDZ binding motif (PDZBM), which binds with PDZ domain-containing proteins, such as protein-tyrosine phosphatase non-receptor type 13 (PTPN13). The HPV16 E6 oncoprotein interacts with the cellular phosphatase and tumor suppressor, PTPN13; this interaction results in degradation of PTPN13. PTPN13 interacts with ephrin B1 which is also a phosphatase substrate. Ephrin B1 is a single pass transmembrane protein ligand that binds and activates cognate Eph receptor tyrosine kinases. In addition, ephrin B1 itself becomes phosphorylated and initiates its own downstream signaling events. In HPV-infected cells, PTPN13 expression is compromised and thus ephrin B1 phosphorylation persists and contributes to an aggressive phenotype and disease progression. Thus, in a further embodiment, the methods further comprise administering to the subject an inhibitor of the interaction between E6 and PTPN13 (E6 binds to PTPN13 at PDZBM #4 of PTPN13). Any suitable inhibitor may be used, including but not limited to peptides that compete with E6 for binding to PTPN13, or that compete with PTPN13 for binding to E6. In another embodiment, the methods may further comprise administering to the subject an inhibitor of ephrin B1 phosphorylation. Any suitable inhibitor may be used, including but not limited to Src kinase inhibitors, including but not limited to dasatanib (chemical structure shown below), or a pharmaceutically acceptable salt thereof.
In another embodiment, the tumor has low PTPN13 expression levels or protein/protein activity levels, such as tumors in which PTPN13 expression levels or protein levels/activity are low due to promoter methylation, mRNA degradation, etc. In this embodiment, tumors with PTPN13 expression or protein level/activity below a threshold level (such as a control of normal levels of PTPN13 expression) are treated with the methods of the disclosure. In this embodiment, ephrin B1 phosphorylation would persist and the methods of the disclosure would be effective for treating such tumors. Exemplary tumor types with low to no PTPN13 expression include, but are not limited to, certain breast cancers (such as triple negative breast cancers: Révillion F, Puech C, Rabenoelina F, Chalbos D, Peyrat J P, Freiss G. Int J Cancer. 2009 Feb. 1; 124(3):638-43; Vermeer P D, Bell M, Lee K, Vermeer D W, Wieking B G, Bilal E, Bhanot G, Drapkin R I, Ganesan S, Klingelhutz A J, Hendriks W J, Lee J H. PLoS One. 2012; 7(1):e30447). See also Science. 2004 May 21; 304(5674):1164-6 in which PTPN13 may be mutated in colorectal, lung, breast, and gastric cancers,
The anti-human ephrin B1 antibody or fragment thereof may be administered by any suitable route, including but not limited to oral, topical, parenteral, intranasal, pulmonary, or rectal administration in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. In one embodiment, the anti-human ephrin B1 antibody or fragment thereof is administered via local delivery, such as by direct injection into or peri-tumorally (i.e.: adjacent to the tumor and contacting the microenvironment surrounding the tumor, both of which will have exosomes that are therapy targets).
The anti-human ephrin B1 antibody or fragment thereof may be administered in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants. The anti-human ephrin B1 antibody or fragment thereof may be administered as the sole therapy, or may be administered in combination with other therapeutic modalities (i.e.: chemotherapy, radiation therapy, surgical removal of the tumor, etc.).
3 rats were each immunized with a DNA vector that contained the extracellular domain of human EphrinB1. Each rat received 4 genetic immunizations (once a week). On day 31, immune serum was collected and tested. Screening consisted of transfection of mammalian cells with the immunization construct containing the ECD of human EphrinB1. Serum from immunized rats was used to see if antibodies bound to the transfected mammalian cells (that had surface expression of EphrinB1's ECD). This was assessed by flow cytometry. Immune sera from all three rats was demonstrated to bind to surface expressed human EphrinB1 ECD. Rats were sacrificed and spleens isolated and fused with myeloma cells to generate the hybridomas. Fused hybridomas were cloned in 96 well dishes and screened by flow cytometry to identify clones that bind the ECD of EphrinB1 the best. 108 clones were generated; of these 54 clones had the highest binding, and 10 clones were selected for further testing. These 10 clones were expanded to T25 dishes and assessed for appropriate activity; three clones were selected for detailed analysis (BXD-1H7-G5, BXD-2G8-D3, and BXD-6C4-B9).
Total RNA was isolated from frozen hybridoma cell lysates in RNAlater following the technical manual of TRIzol® Reagent. Total RNA was then reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit. The antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE). Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes were sequenced for each fragment.
WMG
AMWNGGRTDYNSAFKSRLSISRDTSKSQVFLNMNSLQADDTAKYFCARLDYWGQGVMVTVAS
LLIY
GASNRHTGVPDRFTGSGFGRDFTLTISNVEAEDLAVYYCLHYGSIPFTFGSGTKLEIK.
LEWMG
AMWNGGGTDYNSAFKSRLSISRDTSKSQVFLKMNSLQTDDTAKYFCATPTDWGQGVMVTV
SS
PKLLIY
GASSRHTGVPDRFTGSGFGRDFTLTITNVEAEDLAWYCLHYGSIPFTFGSGTRLEIK
PEWLG
FIRNKVNGYTTDYNPSVKGRFTISRENTQNMLYLQMNTLRAEDTATYYCTREDYYYSGRF
KLLIY
HTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCLQHDSRPRTFGGGTKLDLK.
Our data support a role for exosomal EphrinB1 in potentiating axonogenesis and tumor growth. To test the utility of blocking EphrinB1 for disease control, we generated anti-human EphrinB1 antibodies as described above. We verified antibody binding to surface expressed Ephrin B1 and that the antibodies recognize human but not mouse Ephrin B1 (data not shown). To test the ability of the EphrinB1 antibodies to block neurite outgrowth in vitro, they were incubated with conditioned media from SCC47-EphrinB1 cells, which are SCC47 cells (human HPV positive head and neck squamous cell carcinoma cell line) that over-express human EphrinB1. Twenty-four hours later, the media was used to stimulate PC12 cells, which can be stimulated to differentiate into neuron-like cells, and thus provide a model to screen exosomes to induce neurite outgrowth. The next day. PC12 cells were fixed, stained for beta-III tubulin (a neuron-specific tubulin isoform) and neurite outgrowth quantified. The figure shows that all but one antibody (2G4) antibodies tested were able to significantly attenuate neurite outgrowth relative to that induced by conditioned media from SCC47-EphrinB1 (
To test the utility of the EphrinB1 antibodies to block tumor growth, immune incompetent NOD SCID mice were implanted with human SCC47-EphrinB1 tumors. There were four groups of mice with N=5 mice/group. Two groups of mice served as controls; one control group was injected with vehicle alone while the second control group was injected with isotype matched IgG (IgG2a/2b). The other two groups were each injected with a different EphrinB1 antibody clone (1H7 or 2G8). Tumor growth was followed and mice were sacrificed at day 14 post-tumor implantation. Tumor growth of the control groups were not significantly different from each other while tumors from the EphrinB1 antibody injected groups were significantly smaller than controls (
NSG mice having no immune system were injected with human SCC1 (HPV negative human head and neck squamous cell carcinoma cell line) cells that express endogenous (basal) EphrinB1 or SCC1 cells that stably over-express EphrinB1 (SCC1 OE #18). These animals were treated with 20 μg purified antibody daily by intraperitoneal injection and tumor growth followed. As shown in
For the characterization of Ephrin B1/1H7, Ephrin B1/2G8 and Ephrin B1/6C4 complexes, the measurements were performed using an Autoflex™ II MALDI ToF mass spectrometer (Bruker) equipped with CovalX's HM4 interaction module. 1 μl of the mixture obtained was mixed with 1 μl of a matrix composed of a re-crystallized sinapinic acid matrix (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA 0.1% (K200 MALDI Kit). After mixing, 1 μl of each sample was spotted on the MALDI plate (SCOUT™ 384). After crystallization at room temperature, the plate was introduced in the MALDI mass spectrometer and analyzed immediately. The analysis has been repeated in triplicate
In order to determine the epitope of Ephrin B1/1H7, Ephrin B1/2G8 and Ephrin B1/6C4 complexes with high resolution, the protein complexes were incubated with deuterated cross-linkers and subjected to multi-enzymatic cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high resolution mass spectrometry (nLC-LTQ-Orbitrap™ MS) and the data generated were analyzed using XQuest™ and Stavrox™ software.
Specifically, 20 μL of Ephrin B1/1H7, Ephrin B1/2G8 or Ephrin B1/6C4 mixtures prepared were mixed with 2 μL of DSS d0/d12 (2 mg/mL; DMF) before 180 minutes incubation time at room temperature. After incubation, reaction was stopped by adding 1 μL of Ammonium Bicarbonate (20 mM final concentration) before 1 h incubation time at room temperature. Then, the solution was dried using a SPEEDVAC™ before H2O 8M urea suspension (20 μL). After mixing, 2 μl of DTT (500 mM) were added to the solution. The mixture was then incubated 1 hour at 37° C. After incubation, 2 μl of iodoacetamide (1M) were added before 1 hour incubation time at room temperature, in a dark room. After incubation, 80 μl of the proteolytic buffer were added. The trypsin buffer contains 50 mM Ambic™ pH 8.5, 5% acetonitrile; The Chymotrypsin buffer contains Tris HCl 100 mM, CaCL2 10 mM pH 7.8; The ASP-N buffer contains Phosphate buffer 50 MM pH 7.8; The elastase buffer contains Tris HCl 50 mM pH 8.0 and the thermolysin buffer contains Tris HCl 50 mM, CaCL2 0.5 mM pH 9.0.
Epitope mapping results indicated that the human ephrin B1 epitopes for the antibodies are as follows:
Aligning these sequences against one another demonstrates that all 3 epitopes are within the human ephrin B1 sequence KNLEPVSWSSLNPKFLSGKGLVIYPKIGDKL (SEQ ID NO:32)
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/847,863 filed May 14, 2019, incorporated by reference herein in its entirety
This invention was made with government support under grant number P20GM103548 awarded by the National Institute of General Medical Sciences. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/032707 | 5/13/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62847863 | May 2019 | US |
