U.S. patent application number 15/876402 was filed with the patent office on 2018-10-18 for anti-efna4 antibody-drug conjugates.
This patent application is currently assigned to PFIZER, INC.. The applicant listed for this patent is ABBVIE STEMCENTRX LLC, PFIZER, INC.. Invention is credited to Alexander John BANKOVICH, Marc Isaac DAMELIN, Scott J. DYLLA, Kiran Manohar KHANDKE, Puja SAPRA.
Application Number | 20180296691 15/876402 |
Document ID | / |
Family ID | 51869058 |
Filed Date | 2018-10-18 |
United States Patent
Application |
20180296691 |
Kind Code |
A1 |
DAMELIN; Marc Isaac ; et
al. |
October 18, 2018 |
ANTI-EFNA4 ANTIBODY-DRUG CONJUGATES
Abstract
The present invention provides for anti-EFNA4 antibody-drug
conjugates and methods for preparing and using the same.
Inventors: |
DAMELIN; Marc Isaac; (Park
Ridge, NJ) ; KHANDKE; Kiran Manohar; (Nanuet, NY)
; SAPRA; Puja; (River Edge, NJ) ; BANKOVICH;
Alexander John; (San Francisco, CA) ; DYLLA; Scott
J.; (Emerald Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PFIZER, INC.
ABBVIE STEMCENTRX LLC |
NEW YORK
NORTH CHICAGO |
NY
IL |
US
US |
|
|
Assignee: |
PFIZER, INC.
NEW YORK
NY
ABBVIE STEMCENTRX LLC
NORTH CHICAGO
IL
|
Family ID: |
51869058 |
Appl. No.: |
15/876402 |
Filed: |
January 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15176452 |
Jun 8, 2016 |
9872922 |
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15876402 |
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14525442 |
Oct 28, 2014 |
9381205 |
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15176452 |
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61899800 |
Nov 4, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/34 20130101; C07K 16/28 20130101; C07K 2317/24 20130101;
C07K 2317/77 20130101; A61K 31/704 20130101; A61K 47/6849 20170801;
A61K 47/6851 20170801; A61K 47/6807 20170801; C07K 2317/565
20130101; C07K 2317/76 20130101; C07K 2317/73 20130101; C07K
2317/92 20130101; A61K 2039/505 20130101; C07K 16/30 20130101 |
International
Class: |
A61K 47/68 20170101
A61K047/68; A61K 31/704 20060101 A61K031/704; C07K 16/30 20060101
C07K016/30 |
Claims
1.-69. (canceled)
70. An antibody-drug conjugate of the formula: Ab-(L-D), wherein:
(a) Ab is an antibody, or antigen-binding fragment thereof, that
binds to EFNA4 and comprises: (i) a heavy chain variable region
comprising three CDRs set forth as SEQ ID NOs: 15, 19, and 23 and a
light chain variable region comprising three CDRs set forth as SEQ
ID NOs: 29, 33, and 35; or (ii) a heavy chain variable region
comprising three CDRs set forth as SEQ ID NOs: 41, 45, and 49 and a
light chain variable region comprising three CDRs set forth as SEQ
ID NOs: 55, 59, and 61; and (b) L-D is a linker-drug moiety,
wherein L is a linker and D is a DNA-damaging agent.
71. The antibody-drug conjugate of claim 70, wherein the Ab
comprises: (i) a heavy chain variable region having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 13 and a
light chain variable region having an amino acid sequence that is
at least 90% identical to SEQ ID NO: 27; or (ii) a heavy chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 39 and a light chain variable region having
an amino acid sequence that is at least 90% identical to SEQ ID NO:
53.
72. The antibody-drug conjugate of claim 71, wherein the Ab
comprises: (i) a heavy chain variable region set forth as SEQ ID
NO: 13 and a light chain variable region set forth as SEQ ID NO:
27; or (ii) a heavy chain variable region set forth as SEQ ID NO:
39 and a light chain variable region set forth as SEQ ID NO:
53.
73. The antibody-drug conjugate of claim 72, wherein the Ab
comprises a IgG1 heavy chain constant region.
74. The antibody-drug conjugate of claim 73, wherein the Ab
comprises: (i) a heavy chain set forth as SEQ ID NO: 25; or (ii) a
heavy chain set forth as SEQ ID NO: 51.
75. The antibody-drug conjugate of claim 72, wherein the Ab
comprises a kappa light chain constant region.
76. The antibody-drug conjugate of claim 75, wherein the Ab
comprises: (i) a light chain set forth as SEQ ID NO: 37; or (ii) a
light chain set forth as SEQ ID NO: 63.
77. The antibody-drug conjugate of claim 72, wherein the Ab
comprises: (i) a heavy chain set forth as SEQ ID NO: 25 and a light
chain set forth as SEQ ID NO: 37; or (ii) a heavy chain set forth
as SEQ ID NO: 51 and a light chain set forth as SEQ ID NO: 63.
78. The antibody-drug conjugate of claim 1, having a drug-antibody
ratio (DAR) from 1 to 12.
79. An antibody-drug conjugate of the formula: Ab-(L-D), wherein:
(a) Ab is an antibody, or antigen-binding fragment thereof,
comprising (i) a heavy chain set forth as SEQ ID NO: 25 and a light
chain set forth as SEQ ID NO: 37; or (ii) a heavy chain set forth
as SEQ ID NO: 51 and a light chain set forth as SEQ ID NO: 63; and
(b) L-D is a linker-drug moiety, wherein L is a linker and D is a
DNA-damaging agent.
80. A composition comprising a plurality of antibody-drug
conjugates of the formula: Ab-(L-D), and optionally a
pharmaceutical carrier, wherein: (a) Ab is an antibody, or
antigen-binding fragment thereof, that binds to EFNA4 and comprises
(i) a heavy chain variable region comprising three CDRs set forth
as SEQ ID NOs: 15, 19, and 23 and a light chain variable region
comprising three CDRs set forth as SEQ ID NOs: 29, 33, and 35; or
(ii) a heavy chain variable region comprising three CDRs set forth
as SEQ ID NOs: 41, 45, and 49 and a light chain variable region
comprising three CDRs set forth as SEQ ID NOs: 55, 59, and 61; and
(b) L-D is a linker-drug moiety, wherein L is a linker and D is a
DNA-damaging agent; and wherein the composition has an average DAR
from 1 to 12.
81. The composition of claim 80, wherein the composition has an
average DAR within the range of 3 to 5.
82. The composition of claim 81, wherein the composition has an
average DAR within the range of 3 to 4.
83. The composition of claim 82, wherein the composition has an
average DAR within the range of 4 to 5.
84. The composition of claim 83, wherein the composition has an
average DAR of about 4.
85. The composition of claim 80, wherein at least 50% of the
antibody-drug conjugates have a DAR from 3 to 5.
86. The composition of claim 85, wherein at least 60% of the
antibody-drug conjugates have a DAR from 3 to 5.
87. The composition of claim 86, wherein at least 70% of the
antibody-drug conjugates have a DAR from 3 to 5.
88. The composition of claim 87, wherein the composition has at
least 75% antibody-drug conjugates having a DAR from 3 to 5.
89. The composition of claim 86, wherein the composition has about
70% to 80% antibody-drug conjugates having a DAR from 3 to 5.
Description
RELATED APPLICATIONS
[0001] Priority is claimed to provisional U.S. Application No.
61/899,800, filed 4 Nov. 2013, which is incorporated by reference
herein in its entirety.
SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC72005_Sequence_Listing.txt" created on Nov. 1, 2013, and having
a size of 56 KB. The sequence listing contained in this .txt file
is part of the specification and which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to ephrin-A4 ligand (EFNA4)
antibodies and antibody-drug conjugates. The present invention
further relates to the methods of using such antibodies and
antibody-drug conjugates for the treatment of cancer.
BACKGROUND
[0004] Ephrin receptors (EPH), the largest family of receptor
tyrosine kinases, are type-I transmembrane proteins that bind with
ephrin ligands (EFN). Receptors in the EPH subfamily typically have
a single kinase domain and an extracellular region containing a
Cys-rich domain and 2 fibronectin type III repeats. Based upon
sequence analyses, ephrin ligands can be divided into two groups:
ephrin-A ligands (EFNA) and three ephrin-B ligands (EFNB). EFNA
ligands (i.e., EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNA6) are
typically anchored to the cell surface via glycosyl
phosphatidylinositol (GPI) linkages, although some non-GPI-anchored
proteins are produced through alternative splicing of ephrin mRNAs,
such as EFNA4. EFNB ligands (i.e. EFNB1, EFNB2, EFNB3) contain a
transmembrane domain and a short cytoplasmic region with conserved
tyrosine residues and a PDZ-binding motif. EFNA ligands
preferentially bind with any of the nine different ephrin A
receptors (EPHA) (i.e., EPHA1 , EPHA2, EPHA3, EPHA4, EPHA5, EPHA6,
EPHA7, EPHA8, EPHA9), whereas EFNB ligands preferentially bind with
any of six different ephrin B receptors (EPHB) (i.e. EPHB 1, EPHB2,
EPHB 3, EPHB4, EPHB 5, EPHB 6), although some cross-interactions
have been reported.
[0005] EFN-EPH signaling can be bi-directional (impacting both the
ligand- and receptor-expressing cells) and regulates a broad range
of biological activities including neural development, cell
patterning, angiogenesis, and cell motility and invasion. In the
context of cancer, the expression of various EPHs and EFNs has been
observed, and various functions have been reported (Hafner et al.,
Clinical Chemistry 50(3):490-499, 2004; Surawska et al., Cytokine
& Growth Factor Reviews 15(6):419-433, 2004; Pasquale, E. B,
Nature Reviews Cancer 10(3):165-180, 2010). Due to ligand-receptor
binding promiscuity as well as functional overlap, it has been
difficult to precisely define the roles of each EFN and EPH. While
therapeutic targeting of the EPH receptors for the treatment of
cancer has been explored targeting of EFN ligands has not been
pursued to any great extent (Pasquale, E. B., Nature Reviews Cancer
10(3):165-180, 2010).
[0006] There remains a significant need for additional therapeutic
options for cancers. To this end, the present invention provides
novel antibody-drug conjugates that target EFNA4-positive
cancers.
SUMMARY
[0007] The present invention provides EFNA4 antibody-drug
conjugates and their use in detection, prophylaxis, and therapy of
EFNA4 associated disorders. An EFNA4 antibody-drug conjugate of the
invention is generally of the formula: Ab-(L-D), wherein Ab is an
antibody, or antigen-binding fragment thereof, that binds to EFNA4,
or an EFNA4-binding fragment thereof; and L-D is a linker-drug
moiety, wherein L is a linker, and D is a drug. The Ab of the
disclosed antibody-drug conjugate can be any EFNA4-binding
antibody. In some aspects of the invention, the Ab is a chimeric,
CDR-grafted, humanized, or a recombinant human antibody, or
EFNA4-binding fragment thereof. In some aspects of the invention,
the Ab is an internalizing antibody and/or a neutralizing
antibody.
[0008] The present invention also provides EFNA4 antibody-drug
conjugates and their use in detection, prophylaxis, and therapy of
EFNA4 associated disorders. An EFNA4 antibody-drug conjugate of the
invention is generally of the formula: Ab-(L-D), wherein Ab is an
antibody, or antigen-binding fragment thereof, that binds to EFNA4,
or an EFNA4-binding fragment thereof; and L-D is a linker-drug
moiety, wherein L is a linker, and D is calicheamicin.
[0009] In particular aspects of the invention, the Ab is a huE22 or
hu47 antibody, or an antibody that competes for binding to human
EFNA4 with huE22 or huE47, and/or an antibody that binds to the
same epitope as a huE22 or huE47 antibody. For example, the Ab may
compete for binding to human EFNA4 with, and/or bind the same
epitope as, an antibody comprising (a) a heavy chain variable
region set forth as SEQ ID NO: 13 and a light chain variable region
set forth as SEQ ID NO: 27; or (b) a heavy chain variable region
set forth as SEQ ID NO: 39 and a light chain variable region set
forth as SEQ ID NO: 53.
[0010] Among Abs that compete for binding to human EFNA4 with
huE22, and/or bind to the same epitope as huE22, representative Abs
useful for preparing EFNA4 antibody-drug conjugates of the
invention include antibodies comprising at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one heavy chain variable region comprises
three CDRs defined by SEQ ID NOs: 15, 19, and 23. Additional Abs
include antibodies comprising at least one heavy chain variable
region and at least one light chain variable region, wherein the at
least one light chain variable region comprises three CDRs defined
as SEQ ID NOs: 29, 33, and 35. Additional Abs include antibodies
comprising (a) a heavy chain variable region comprising three CDRs
set forth as SEQ ID NOs: 15, 19, and 23; and (b) a light chain
variable region comprising three CDRs set forth as SEQ ID NOs: 29,
33, and 35.
[0011] In other EFNA4 antibody-drug conjugates of the invention,
the Ab comprises a heavy chain variable region having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 13 and a
light chain variable having an amino acid sequence that is at least
90% identical to SEQ ID NO: 27, for example, a heavy chain variable
region set forth as SEQ ID NO: 13 and a light chain variable region
set forth as SEQ ID NO: 27.
[0012] Among Abs that compete for binding to human EFNA4 with
huE47, and/or bind to the same epitope as huE47, representative Abs
useful for preparing EFNA4 antibody-drug conjugates of the
invention include antibodies comprising at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one heavy chain variable region comprises
three CDRs defined by SEQ ID NOs: 41, 45, and 49. Additional Abs
include antibodies comprising at least one heavy chain variable
region and at least one light chain variable region, wherein the at
least one light chain variable region comprises three CDRs defined
as SEQ ID NOs: 55, 59, and 61. Additional Abs include antibodies
comprising (a) a heavy chain variable region comprising three CDRs
set forth as SEQ ID NOs: 41, 45, and 49; and (b) a light chain
variable region comprising three CDRs set forth as SEQ ID NOs: 55,
59, and 61.
[0013] In other EFNA4 antibody-drug conjugates of the invention,
the Ab comprises a heavy chain variable region having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 39 and a
light chain variable having an amino acid sequence that is at least
90% identical to SEQ ID NO: 53, for example, a heavy chain variable
region set forth as SEQ ID NO: 39 and a light chain variable region
set forth as SEQ ID NO: 53.
[0014] In some aspects of the invention, EFNA4 antibody-drug
conjugates comprise an Ab comprising a IgG1 heavy chain constant
region, a kappa light chain constant region, or a IgG1 heavy chain
constant region and a kappa light chain constant region. For
example, Abs useful for preparing EFNA4 antibody-drug conjugates of
the invention include antibodies comprising a heavy chain set forth
as SEQ ID NO: 25, a light chain set forth as SEQ ID NO: 37, or a
heavy chain set forth as SEQ ID NO: 25 and a light chain set forth
as SEQ ID NO: 37. Additional examples include antibodies comprising
a heavy chain set forth as SEQ ID NO: 51, a light chain set forth
as SEQ ID NO: 63, or a heavy chain set forth as SEQ ID NO: 51 and a
light chain set forth as SEQ ID NO: 63.
[0015] In other aspects of the invention, an EFNA4 antibody-drug
conjugate of the invention comprises an antibody having a heavy
chain variable region set forth as SEQ ID NO: 13 or 39. In other
aspects of the invention, an EFNA4 antibody-drug conjugate of the
invention comprises an antibody having light chain variable region
set forth as SEQ ID NO: 27 or 53.
[0016] In particular aspects of the invention, the Ab is a huE5 or
hu15 antibody, or an antibody that competes for binding to human
EFNA4 with huE5 or huE15, and/or an antibody that binds to the same
epitope as a huE5 or huE15 antibody. For example, the Ab may
compete for binding to human EFNA4 with, and/or bind the same
epitope as, an antibody comprising (a) a heavy chain variable
region set forth as SEQ ID NO: 5 and a light chain variable region
set forth as SEQ ID NO: 7; or (b) a heavy chain variable region set
forth as SEQ ID NO: 9 and a light chain variable region set forth
as SEQ ID NO: 11.
[0017] In another aspect of the invention, the Ab is a humanized
antibody such as huE5, huE15, huE22 or hu47 antibody,
[0018] Any of the EFNA4 antibody-drug conjugates disclosed herein
may be prepared with a linker comprising
4-(4'acetylphenoxy)butanoic acid (AcBut).
[0019] Any of the EFNA4 antibody drug conjugates disclosed herein
may be prepared with a calicheamicin drug, including N-acetyl
derivatives of calicheamicin, such as
N-acetyl-.gamma.-calicheamicin and N-acetyl-y-calicheamicin
dimethyl hydrazide (CM).
[0020] Any of the EFNA4 antibody-drug conjugates disclosed herein
may have a drug-to-antibody ratio (DAR) from 1 to 12. In a
particular aspect of the invention, an EFNA4 antibody-drug
conjugate of the formula Ab-(L-D) comprises (a) an antibody, or
antigen-binding fragment thereof, Ab, comprising a heavy chain set
forth as SEQ ID NO: 25 and a light chain set forth as SEQ ID NO:
37; and (b) a linker-drug moiety, L-D, wherein L is a linker, and D
is a drug, wherein the linker is 4-(4'acetylphenoxy)butanoic acid
(AcBut), and wherein the drug is N-acetyl-.gamma.-calicheamicin
dimethyl hydrazide (CM).
[0021] In another aspect of the invention, an EFNA4 antibody-drug
conjugate of the formula Ab-(L-D) comprises (a) an antibody, or
antigen-binding fragment thereof, Ab, comprising a heavy chain set
forth as SEQ ID NO: 51 and a light chain set forth as SEQ ID NO:
63; and (b) a linker-drug moiety, L-D, wherein L is a linker, and D
is a drug, wherein the linker is 4-(4'acetylphenoxy)butanoic acid
(AcBut), and wherein the drug is N-acetyl-.gamma.-calicheamicin
dimethyl hydrazide (CM).
[0022] The present invention provides for compositions comprising a
plurality of antibody-drug conjugate disclosed herein and
optionally a pharmaceutical carrier, wherein the composition has an
average DAR within a range of 1 to 12. In a particular aspect of
the invention, the composition may have an average DAR within the
range of 3 to 5. In another aspect of the invention, the
composition may have an average DAR within the range of 3 to 4. In
another aspect of the invention, the composition may have an
average DAR within the range of 4 to 5. In another aspect of the
invention, the composition may have an average DAR of about 4.
[0023] The present invention further provides for a composition
comprising a plurality of an antibody-drug conjugate disclosed
herein and optionally a pharmaceutical carrier, wherein the
composition has at least 50% antibody-drug conjugates having a DAR
from 3 to 5. In another aspect of the invention, the composition
has at least 60% antibody-drug conjugates having a DAR from 3 to 5.
In another aspect of the invention, the composition has at least
70% antibody-drug conjugates having a DAR from 3 to 5. In another
aspect of the invention, the composition has at least 75%
antibody-drug conjugates having a DAR from 3 to 5. In another
aspect of the invention, the composition has about 70% to 80%
antibody-drug conjugates having a DAR from 3 to 5.
[0024] The present invention further provides for an EFNA4
antibody-drug conjugate that is generally of the formula: Ab-(L-D),
wherein Ab is an antibody, or antigen-binding fragment thereof,
that binds to EFNA4, or an EFNA4-binding fragment thereof; and L-D
is a linker-drug moiety, wherein L is vc or mc, and D is a
drug.
[0025] The present invention further provides for an EFNA4
antibody-drug conjugate that is generally of the formula: Ab-(L-D),
wherein Ab is an antibody, or antigen-binding fragment thereof,
that binds to EFNA4, or an EFNA4-binding fragment thereof; and L-D
is a linker-drug moiety, wherein L is a linker, and D is 0101 or
8261.
[0026] The present invention further provides methods for preparing
an EFNA4 antibody-drug conjugate disclosed herein. For example, a
process for producing an antibody-drug conjugate can include the
steps of (a) linking the linker to the drug moiety; (b) conjugating
the linker-drug moiety to the antibody; and (c) purifying the
antibody-drug conjugate.
[0027] Another aspect of the invention includes methods of making,
methods of preparing, methods of synthesis, methods of conjugation,
and methods of purification of the antibody-drug conjugates
disclosed herein, and the intermediates for the preparation,
synthesis, and conjugation of the antibody-drug conjugates
disclosed herein.
[0028] Further provided are pharmaceutical compositions comprising
an EFNA4 antibody-drug conjugate disclosed herein and a
pharmaceutically acceptable carrier.
[0029] In other aspects are provided methods of treating an EFNA
associated disorder by administering a therapeutically effective
amount of a composition comprising an EFNA4 antibody-drug conjugate
disclosed herein. Representative EFNA associated disorders include
hyperproliferative disorders, such as neoplastic disorders, such as
solid tumors (e.g., breast cancer, ovarian cancer, colorectal
cancer, liver cancer, lung cancer, etc.) and hematologic
malignancies (e.g., leukemia, etc.). Also provided are uses of the
disclosed EFNA4 antibody-drug conjugates for the manufacture of a
medicament for treating an EFNA associated disorder in a subject.
Also provided are EFNA4 antibody-drug conjugates for use in the
treatment of an EFNA associated disorder.
[0030] In other aspects, the present invention provides for methods
of treating an EFNA associated disorder in a subject by
administering a therapeutically effective amount of a composition
comprising an EFNA4 antibody-drug conjugate disclosed herein and a
chemotherapeutic agent.
[0031] Another aspect of the invention includes methods of treating
a disorder characterized by the overexpression of EFNA4 in a
patient with an antibody-drug conjugate disclosed herein. In other
aspects, the present invention provides for methods of treating
cancer characterized by the overexpression of EFNA4 in a patient
with an antibody-drug conjugate disclosed herein.
[0032] In still other aspects, the present invention provides a
method of reducing tumor initiating cells in a tumor cell
population. For example, the method can comprise contacting a tumor
cell population, wherein the population comprises tumor initiating
cells and tumor cells other than tumor initiating cells, with an
EFNA4 antibody-drug conjugate; whereby the frequency of tumor
initiating cells in the tumor cell population is reduced. The
contacting step may be performed in vitro or in vivo.
[0033] Another aspect of the invention includes diagnostic and
therapeutic uses for the compounds and compositions disclosed
herein.
[0034] Other aspects of the invention include articles of
manufacture, i.e. kits, comprising an antibody-drug conjugate
disclosed herein, a container, and a package insert or label
indicating a treatment.
[0035] These and other aspects of the invention will be appreciated
by a review of the application as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 provides an alignment of human EFNA4 a, b and c
isoform sequences showing amino acid differences (SEQ ID NOS:
2-4).
[0037] FIG. 2 provides the genetic arrangement and the heavy and
light chain CDR sequences (derived from an analysis of the VBASE2
database) of EFNA4 antibodies.
[0038] FIGS. 3A and 3B provide binding data for huE22 and huE47
antibodies, respectively, to illustrate binding specificity for
EFNA4.
[0039] FIGS. 4A and 4B provide the structure of AcBut-CM and
AcBut-CM conjugated to an antibody (X), respectively.
[0040] FIG. 5 provides the hydrophobic interaction chromatography
(HIC) analysis of huE22-AcBut-CM.
[0041] FIG. 6 provides an UPLC-HIC analysis of purified
huE22-AcBut-CM on TSKgel Butyl-NPR for the presence of unconjugated
antibody.
[0042] FIG. 7 provides an UPLC-SEC analysis of purified
huE22-AcBut-CM on BEH200 SEC for the presence of aggregates and
dimers.
[0043] FIG. 8 provides a reverse phase analysis of purified
huE22-AcBut-CM on Zorbax 300SB-CN for presence of free drug.
[0044] FIG. 9 provides a cIEF analysis of purified huE22-AcBut-CM
on iCE280 for the DAR distribution profile.
[0045] FIG. 10 provides the effect of multiple freeze-thaws on
stability of huE22-AcBut-CM.
[0046] FIG. 11 shows the efficacy of huE22-AcBut-CM in the Breast-5
(BR5) triple-negative breast cancer (TNBC) PDX.
[0047] FIG. 12 shows the efficacy of huE22-AcBut-CM in the
Breast-13 (BR13) TNBC PDX.
[0048] FIG. 13 shows the efficacy of huE22-AcBut-CM in the
Breast-22 (BR22) PDX.
[0049] FIG. 14 shows the efficacy of huE22-AcBut-CM in the
Breast-31 (BR31) TNBC PDX.
[0050] FIG. 15 shows the efficacy of huE22-AcBut-CM in the
Ovarian-45 (OV45) ovarian cancer PDX.
[0051] FIG. 16 shows the efficacy of huE22-AcBut-CM in the
Ovarian-55 (OV55) ovarian cancer PDX.
[0052] FIG. 17 shows the efficacy of huE22-AcBut-CM in the
Ovarian-44 (OV44) ovarian cancer PDX.
[0053] FIG. 18 shows the efficacy of huE22-AcBut-CM in the
Ovarian-63 (OV63) ovarian cancer PDX.
[0054] FIG. 19 shows the efficacy of huE15, huE22 and huE47
conjugated to the microtubule inhibitors (MTIs) vc0101 and mc8261
in the BR22 TNBC PDX.
[0055] FIG. 20 shows the efficacy of huE15, huE22 and huE47
conjugated to the MTI vc0101 in the BR31 TNBC PDX.
[0056] FIG. 21 shows the in vitro cytotoxicity of huE22-AcBut-CM in
EFNA4-expressing 293T cells (open diamonds) versus control-AcBut-CM
ADC (diagonal-hatched circles).
[0057] FIG. 22 shows immunohistochemistry with anti-hIgG1 antibody
(diagonal-hatched circles) and anti-.gamma.-H2A.X antibody (open
diamonds) of BR5 TNBC PDX tumors exposed to huE22-AcBut-CM.
[0058] FIG. 23 shows tumor growth curves for mice implanted with
ESA.sup.+CD46.sup.+CD324.sup.+ (diagonal-hatched circles) or
ESA.sup.+CD46.sup.+CD324.sup.- (open circles) cells isolated from
dissociated BR22 TNBC PDX tumors.
[0059] FIG. 24 shows tumor growth curves for mice implanted with
ESA.sup.+CD46.sup.+CD324.sup.+ (diagonal-hatched circles) or
ESA.sup.+CD46.sup.+CD324.sup.- (open circles) cells isolated from
dissociated BR31 TNBC PDX tumors.
[0060] FIG. 25 shows expression of EFNA4 mRNA in normal-adjacent
breast, TNBC, non-TNBC breast using TOGA data.
[0061] FIG. 26 shows EFNA4 copy number in breast cancer tumor
samples from the TOGA and METABRIC datasets.
[0062] FIG. 27 shows EFNA4 copy number in ovarian cancer tumor
samples from the TOGA and METABRIC datasets.
[0063] FIG. 28 shows EFNA4 copy number in hepatocellular carcinoma
(HCC) tumor samples from the TOGA and METABRIC datasets.
[0064] FIG. 29 shows a comparison of analytical HICs for purified
huE22-AcBut-CM ADCs generated from a ratio of CM to huE22 Ab of 4
m/m and 6 m/m.
[0065] FIG. 30 shows a further comparison of analytical HICs for
purified huE22-AcBut-CM ADCs generated from a ratio of CM to huE22
Ab of 4 m/m and 6 m/m.
DETAILED DESCRIPTION OF THE INVENTION
[0066] The present invention provides antibody-drug conjugates that
bind to ephrin-A ligands (or EFNA), such as EFNA4. The invention
also provides processes for preparing the conjugates using EFNA4
antibodies, linkers, and drugs. The antibody-drug conjugates of the
invention are useful for the preparation and manufacture of
compositions, such as medicaments that may be used in the
diagnosis, prophylaxis, and/or treatment of hyperproliferative
disorders characterized by EFNA4 expression. In some aspects of the
invention, the disclosed antibody-drug conjugates may reduce the
frequency of tumor initiating cells (TIC), which encompass both
tumor perpetuating cells (TPC) and highly proliferative tumor
progenitor cells (TProg).
I. EFNA Physiology
[0067] PCT International Publication No. WO2012/118547 describes
that, as with all cell surface receptor-ligand interactions,
engagement of the ephrin receptor (EPH) by an ephrin ligand (EFN)
ultimately results in the activation of intracellular signaling
cascades. Although receptor-ligand interactions may take place
between molecules on the surface of the same cell (cis
interactions), it is generally thought that cis interactions do not
lead to the triggering of signaling cascades, or that cis
interactions may actually antagonize signaling cascades initiated
by trans interactions (e.g., between receptors and ligands on
separate cells). One aspect of EPH-EFN trans interactions is the
capacity for the triggering of two signaling cascades upon
receptor-ligand engagement--a forward signaling cascade in the cell
expressing the EPH, and a reverse signaling cascade in the cell
expressing the EFN. The activation of two separate signaling
cascades may reflect cell sorting and cell positioning processes
that EPH and EFN have evolved to co-ordinate in animal embryonic
development.
[0068] EPH-EFN signaling frequently activates cell-signaling
pathways that regulate cytoskeletal dynamics and lead to modulation
of the adhesive and repulsive interactions between different types
of cells. Generally, EPH and EFN proteins are found at much higher
levels during embryogenesis versus those observed in adult tissues,
although continued low-level expression in the adult may reflect
roles for these molecules in the normal function of tissues such as
the adult gut, which has a well-defined architecture arising from
the migration of differentiating cells from their source at the
tissue stem cell in the crypt to their final location at the
surface of the villi facing the intestinal lumen. Since EPH were
first identified in hepatocellular carcinomas, and EPH and EFN
expression is typically limited in adults, reactivation of the
expression of EFN and/or EPH in human cancers may be linked to the
dedifferentiation of the cancer cells and/or the ability of these
cancer cells to invade surrounding normal tissue and to migrate
from the site of the primary tumor to distant locations. Other
studies have suggested that EPH-EFN interactions also have a role
in neoangiogenesis.
[0069] Consistent with findings that EPH-EFN interactions in
non-lymphoid tissues regulate cellular interactions by generating
adhesive or repulsive forces between cells through integrin and
cytoskeleton rearrangements, EPH and EFN molecules found on
lymphoid cells have been shown to mediate cell adhesion to
extracellular matrix components, chemotaxis and cell migration. For
example, EFNA1, (which binds to the EPHA2 receptor and comprises,
for example, an amino sequence as in Genbank accession NM_004428)
engagement on primary CD4 and CD8 T cells has been found to
stimulate cell migration and enhance chemotaxis. Like EFNA1, EFNA4
is expressed on primary CD4 T cells but, due to the promiscuity of
the EPH-EFN interaction, it is unclear if EFNA4 engagement has
similar effects on these cells. However, it has been demonstrated
that mature human B-lymphocytes express EFNA4 and secrete it upon
activation. Further EFNA4, unlike any other EFN or EPH molecule, is
also consistently expressed on or by B cells of chronic lymphocytic
leukemia (CLL) patients. Interestingly, the expression of EFNA4
isoforms as measured by Q-PCR may be correlated with the clinical
manifestation of the disorder. Also, B cells from CLL patients
known to have increased expression of EFNA4 showed impairment in
transendothelial migration potential compared to B cells from
healthy individuals. Evidently, engagement of EFNA4 reduced the
ability of CLL cells to adhere to extracellular matrix molecules
and reduced their chemotactic response to CCL1. Together these
reports suggest a role for EFNA4 in B and T cell trafficking and,
when viewed in combination with the intracellular signaling data
discussed above, make EFNA, and EFNA4 in particular, very
intriguing targets for the development of anti-cancer
therapeutics.
[0070] In addition, the expression of EFNA4 is elevated in various
cancer stem cell populations. Along with concomitant upregulation
of several EPHA receptors in the bulk tumor, this raises the
possibility that EFNA4 mediated ligand-receptor interactions may be
triggering cell signaling cascades linked to tumor proliferation,
neoangiogenesis and/or tumor metastasis. While not wishing to be
bound by any particular theory, It is believed that EFNA4
antibodies and EFNA4 antibody-drug conjugates of the present
invention act, at least in part, by either reducing or eliminating
tumor initiating cell (TIC) frequency thereby interfering with
tumor propagation or survival in a different manner than
traditional standard of care (SOC) therapeutic regimens (e.g.
doxorubicin and irinotecan), or through immunotherapeutic signaling
or delivering a payload able to kill EFNA4 expressing cells. See
Examples 8 and 9.
[0071] Representative EFNA4 protein orthologs include, but are not
limited to, human (NP_005218, NP_872631 or NP_872632), mouse
(NP_031936), chimpanzee (XP_001153095, XP_001152971, XP_524893, and
XP_001152916) and rat (NP_001101162). The transcribed human EFNA4
gene includes at minimum 5817 bp from chromosome 1.
[0072] Three human EFNA4 mRNA transcript variants and encoded
proteins are shown in Table 1, each of which arises from
alternative splicing of the transcribed RNA: (1) a 1276 base pair
variant (NM_005227; EFNA4 transcript variant 1; SEQ ID NO: 1) which
encodes a 201 amino acid proprotein (NP_005218; EFNA4 isoform a;
SEQ ID NO: 2); (2) a 1110 base pair variant (NM_182689; EFNA4
transcript variant 2) which encodes a 207 amino acid proprotein
(NP_872631; EFNA4 isoform b; SEQ ID NO: 3); and (3) a 1111 base
pair variant (NM_182690; EFNA4 transcript variant 3) which encodes
a 193 amino acid proprotein (NP_872632; EFNA4 isoform c; SEQ ID NO:
4).
TABLE-US-00001 TABLE 1 Human EFNA4 mRNA transcript variants and
encoded proteins. SEQ ID NO Sequence 1
CTTCCCTCTTCACTTTGTACCTTTCTCTCCTCGACTGTGAAG
CGGGCCGGGACCTGCCAGGCCAGACCAAACCGGACCTCGGGG
GCGATGCGGCTGCTGCCCCTGCTGCGGACTGTCCTCTGGGCC
GCGTTCCTCGGCTCCCCTCTGCGCGGGGGCTCCAGCCTCCGC
CACGTAGTCTACTGGAACTCCAGTAACCCCAGGTTGCTTCGA
GGAGACGCCGTGGTGGAGCTGGGCCTCAACGATTACCTAGAC
ATTGTCTGCCCCCACTACGAAGGCCCAGGGCCCCCTGAGGGC
CCCGAGACGTTTGCTTTGTACATGGTGGACTGGCCAGGCTAT
GAGTCCTGCCAGGCAGAGGGCCCCCGGGCCTACAAGCGCTGG
GTGTGCTCCCTGCCCTTTGGCCATGTTCAATTCTCAGAGAAG
ATTCAGCGCTTCACACCCTTCTCCCTCGGCTTTGAGTTCTTA
CCTGGAGAGACTTACTACTACATCTCGGTGCCCACTCCAGAG
AGTTCTGGCCAGTGCTTGAGGCTCCAGGTGTCTGTCTGCTGC
AAGGAGAGGAAGTCTGAGTCAGCCCATCCTGTTGGGAGCCCT
GGAGAGAGTGGCACATCAGGGTGGCGAGGGGGGGACACTCCC
AGCCCCCTCTGTCTCTTGCTATTACTGCTGCTTCTGATTCTT
CGTCTTCTGCGAATTCTGTGAGCCAAGCAGACCTTCCCTCTC
ATCCCAAGGAGCCAGAGTCCTCCCAAGATCCCCTGGAGGAGG
AGGGATCCCTGCTGCCTGCACTGGGGGTGCCAATTCAGACCG
ACAAGATGGAGCATTGATGGGGGAGATCAGAGGGTCTGAGGT
GACTCTTGCAGGAGCCTGTCCCCTCATCACAGGCTAAAGAAG
AGCAGTAGACAGCCCTGGACACTCTGAAGCAGAGGCAAGACA
AACACAGGCGCTTTGCAGGCTGCTCTGAGGGTCTCAGCCCAT
CCCCCAGGAGGACTGGGATTTGGTATGATCAAATCCTCAAGC
CAGCTGGGGGCCCAGGCTGAAGACCTGGGGACAGGTCGATTG
CTGGACCAGGGCAAAGAAGAAGCCCTGCCATCTGTGCCCTGT
GGGCCTTTTCCCTGGGGCAGCACCTTGCCCTCCCCAGGGGAT
CACTCACTTGTCTTCTATGAAGACGGACTCTTCATGAGGTTG
AATTTCATGCCAGTTTGTATTTTTATAAGTATCTAGACCAAA
CCTTCAATAAACCACTCATCTTTTTGTTGCCCTCCCCAAAAA AAAAAAAAAAAAAAAA 2
MRLLPLLRTVLWAAFLGSPLRGGSSLRHVVYWNSSNPRLLRG
DAVVELGLNDYLDIVCPHYEGPGPPEGPETFALYMVDWPGYE
SCQAEGPRAYKRWVCSLPFGHVQFSEKIQRFTPFSLGFEFLP
GETYYYISVPTPESSGQCLRLQVSVCCKERKSESAHPVGSPG
ESGTSGWRGGDTPSPLCLLLLLLLLILRLLRIL 3
MRLLPLLRTVLWAAFLGSPLRGGSSLRHVVYWNSSNPRLLRG
DAVVELGLNDYLDIVCPHYEGPGPPEGPETFALYMVDWPGYE
SCQAEGPRAYKRWVCSLPFGHVQFSEKIQRFTPFSLGFEFLP
GETYYYISVPTPESSGQCLRLQVSVCCKERRARVLPRSPGGG
GIPAACTGGANSDRQDGALMGEIRGSEVTLAGACPLITG 4
MRLLPLLRTVLWAAFLGSPLRGGSSLRHVVYWNSSNPRLLRG
DAVVELGLNDYLDIVCPHYEGPGPPEGPETFALYMVDWPGYE
SCQAEGPRAYKRWVCSLPFGHVQFSEKIQRFTPFSLGFEFLP
GETYYYISVPTPESSGQCLRLQVSVCCKERNLPSHPKEPESS
QDPLEEEGSLLPALGVPIQTDKMEH
[0073] It will be appreciated that each of the human EFNA4 proteins
include a predicted signal or leader sequence comprising amino
acids 1-25 of SEQ ID NO: 2 which is clipped off to provide the
mature form of the protein (i.e. 168-182 aa in length). This signal
peptide targets the polypeptide to the cell surface/secretory
pathway. The term "signal sequence," also called signal peptide,
leader peptide, refers to a segment of about 15 to 30 amino acids
at the N terminus of a protein that enables the protein to be
secreted (pass through a cell membrane). The signal sequence is
removed as the protein is secreted. Due to the alternative splicing
of the mRNA with consequent effects upon the protein coding
sequences, the protein isoforms are processed differently by the
cell. Isoform a is membrane localized and anchored to the cell
surface by a glycosylphosphatidylinositol (GPI) linkage, whereas
isoforms b and c lack the GPI-anchor signal sequence and therefore
are expected to be secreted by the cell. An alignment of the three
protein isoforms of human EFNA4 is shown in FIG. 1. As previously
indicated, unless otherwise indicated by direct reference or
contextual necessity the term EFNA4 shall be directed to isoform a
of human EFNA4 and immunoreactive equivalents. It will further be
appreciated that the term may also refer to a derivative or
fragment of a native or variant form of EFNA4 that contains an
epitope to which an antibody or immunoreactive fragment can
specifically bind.
II. EFNA4 Antibody-Drug Conjugates
[0074] The present invention provides antibody-drug conjugates of
the formula Ab-(L-D), wherein (a) Ab is an antibody, or
antigen-binding fragment thereof, that binds to EFNA4, and (b) L-D
is a linker-drug moiety, wherein L is a linker, and D is a drug. In
contrast to the TKIs developed to inhibit Eph receptor signaling,
antibody-drug conjugates (ADCs), such as anti-EFNA4 ADCs, can
target specific surface molecules and the cells expressing them
regardless of their signaling function, as long as the molecules
efficiently internalize. Also provided are methods of preparing and
manufacturing such antibody-drug conjugates, and use of the same in
clinical applications. "Antibody-drug conjugate" or "ADC" refers to
antibodies, or antigen-binding fragments thereof, including
antibody derivatives that bind to EFNA4 and are conjugated to a
drug such as a cytotoxic, cytostatic, and/or therapeutic agent, as
described further herein below. For example, a cytotoxic agent can
be linked or conjugated to an anti-EFNA4 antibody as described
herein for targeted local delivery of the cytotoxic agent to tumors
(e.g., EFNA4 expressing tumor).
[0075] As used herein, the term "EFNA4" includes variants,
isoforms, homologs, orthologs and paralogs. EFNA4 is also known in
the art as ephrin-A4, ephrin-A4 ligand, EPH-related receptor
tyrosine kinase ligand 4, Ligand Of Eph-Related Kinase 4, EFL4,
EPLG4 and LERK4. In some aspects of the invention, antibodies and
antibody-drug conjugates cross-react with EFNA4 from species other
than human, such as EFNA4 of mouse, rat, or primate, as well as
different forms of EFNA4 (e.g., glycosylated EFNA4). In other
aspects, the antibodies and antibody-drug conjugates may be
completely specific for human EFNA4 and may not exhibit species or
other types of cross-reactivity. As used herein the term EFNA4
refers to naturally occurring human EFNA4 unless contextually
dictated otherwise. Therefore, an "EFNA4 antibody", "anti-EFNA4
antibody", "ephrin-A4 antibody" or "ephrin-A4 ligand antibody" or
other similar designation, means any antibody (as defined herein)
that associates, binds or reacts with the EFNA4 type ligand or
isoform, or fragment or derivative thereof. Further, an "EFNA4
antibody-drug conjugate", "anti-EFNA4 antibody-drug conjugate",
"ephrin-A4 antibody-drug conjugate" or "ephrin-A4 ligand
antibody-drug conjugate" means any antibody-drug conjugate or ADC
(as defined herein) that associates, binds or reacts with the EFNA4
type ligand or isoform, or fragment or derivative thereof.
[0076] "Linker (L)" describes the direct or indirect linkage of the
antibody to the drug. Attachment of a linker to an antibody can be
accomplished in a variety of ways, such as through surface lysines,
reductive-coupling to oxidized carbohydrates, and through cysteine
residues liberated by reducing interchain disulfide linkages. A
variety of ADC linkage systems are known in the art, including
hydrazone-, disulfide- and peptide-based linkages.
[0077] "Drug (D)" is any substance having biological or detectable
activity, for example, therapeutic agents, detectable labels,
binding agents, etc., and prodrugs, which are metabolized to an
active agent in vivo. The terms drug, payload and compound are used
interchangeably.
[0078] "L-D" is a linker-drug moiety resulting from a drug (D)
linked to a linker (L).
[0079] Additional scientific and technical terms used in connection
with the present invention, unless indicated otherwise herein,
shall have the meanings that are commonly understood by those of
ordinary skill in the art. Further, unless otherwise required by
context, singular terms shall include pluralities and plural terms
shall include the singular. Generally, nomenclature used in
connection with, and techniques of, cell and tissue culture,
molecular biology, immunology, microbiology, genetics and protein
and nucleic acid chemistry and hybridization described herein are
those well-known and commonly used in the art.
[0080] In particular aspects of the invention, an EFNA4
antibody-drug conjugate of the formula Ab-(L-D) includes (a) an
antibody (Ab), or antigen-binding fragment thereof, including a
heavy chain variable region set forth as SEQ ID NO: 13 and a light
chain variable region set forth as SEQ ID NO: 27; and (b) a
linker-drug moiety (L-D), wherein L is a linker, and D is a drug,
wherein the linker is 4-(4'acetylphenoxy)butanoic acid (AcBut), and
wherein the drug is N-acetyl-.gamma.-calicheamicin dimethyl
hydrazide (CM). In other aspects of the invention, an EFNA4
antibody-drug conjugate of the formula Ab-(L-D) includes (a) an
antibody (Ab), or antigen-binding fragment thereof, having a heavy
chain variable region set forth as SEQ ID NO: 39 and a light chain
variable region set forth as SEQ ID NO: 53; and (b) a linker-drug
moiety (L-D), wherein L is a linker, and D is a drug, wherein the
linker is 4-(4'acetylphenoxy)butanoic acid (AcBut), and wherein the
drug is N-acetyl-y-calicheamicin dimethyl hydrazide (CM).
[0081] In particular aspects of the invention, an EFNA4
antibody-drug conjugate of the formula Ab-(L-D) includes (a) an
antibody (Ab), or antigen-binding fragment thereof, including a
heavy chain set forth as SEQ ID NO: 25 and a light chain set forth
as SEQ ID NO: 37; and (b) a linker-drug moiety (L-D), wherein L is
a linker, and D is a drug, wherein the linker is
4-(4'acetylphenoxy)butanoic acid (AcBut), and wherein the drug is
N-acetyl-.gamma.-calicheamicin dimethyl hydrazide (CM). In other
aspects of the invention, an EFNA4 antibody-drug conjugate of the
formula Ab-(L-D) includes (a) an antibody (Ab), or antigen-binding
fragment thereof, having a heavy chain set forth as SEQ ID NO: 51
and a light chain set forth as SEQ ID NO: 63; and (b) a linker-drug
moiety (L-D), wherein L is a linker, and D is a drug, wherein the
linker is 4-(4'acetylphenoxy)butanoic acid (AcBut), and wherein the
drug is N-acetyl-y-calicheamicin dimethyl hydrazide (CM).
[0082] The present invention further provides antibody-drug
conjugates that have an optimized average DAR and narrow DAR
distribution. See further description below in section IIC Drugs.
The average DAR and DAR distribution can have an effect on the
clinical efficacy of an ADC, in particular, an effect on both
potency and potential toxicity of the ADC, and can have a
significant effect on properties, such as stability and aggregation
of the ADC.
[0083] The DAR (drug-to-antibody ratio) or drug loading, indicating
the number of drug molecules conjugated per antibody, may be from 1
to 12. Compositions, batches, and/or formulations of a plurality of
antibody-drug conjugates may be characterized by an average DAR.
DAR and average DAR can be determined by various conventional means
such as UV spectroscopy, mass spectroscopy, ELISA assay,
radiometric methods, hydrophobic interaction chromatography (HIC),
electrophoresis and HPLC.
[0084] The DAR distribution provides the percent or fraction of
various ADC species, e.g. DAR 1 to 12, that may be present in a
composition, batch, and/or formulation of ADCs. The DAR
distribution of a composition, batch, and/or formulation of ADCs
may be determined by methods known in the art, such as capillary
iso-electric focusing (cIEF). The DAR distribution of a
composition, batch, and/or formulation of ADCs may be characterized
as a highly heterogeneous mixture with a broad DAR distribution,
generally containing a broad range of ADC species with DAR 1 to 12.
The DAR distribution of a composition, batch, and/or formulation of
ADCs may be characterized as a highly homogeneous mixture with a
narrow DAR distribution, generally containing a narrow range of ADC
species having a particular DAR, such as DAR 3 to 5.
[0085] In particular aspects of the present invention, the improved
conjugation and purification conditions disclosed herein provide
anti-EFNA4 ADCs with an optimized average DAR in the range of about
3 to 5, preferably about 4, and a narrow DAR distribution, for
example, less heterogeneity, in which species with a DAR from 3 to
5 (which are the most desired) make up at least 60%, or at least
70%, or about 70% to 80%, or preferably about 75% to 80% of the
total anti-EFNA4 ADC. See FIG. 9.
[0086] In particular aspects of the invention, during conjugation
and purification, it is beneficial to eliminate ADCs having a high
DAR (DAR>5) which are more hydrophobic and demonstrate faster
clearance, which may contribute to higher toxicity and lower the
therapeutic index (TI). In other aspects of the invention, it is
beneficial to eliminate ADCs having a low DAR (DAR<2) which
contribute little to efficacy, however, provide an increase in the
amounts of unconjugated antibody which may compete with the ADC for
the target antigen, such as EFNA4, and lead to a lower TI. In
another aspect of the invention, it is beneficial to eliminate ADCs
having a high DAR (DAR>5) and ADCs having a low DAR
(DAR<2).
[0087] In particular aspects of the invention, the CM to huE22
ratio used in the preparation of a conjugation reaction mixture may
be 4-5 to 1, compared to higher ratios, to generate anti-EFNA4 ADCs
having an optimized DAR and eliminate higher DAR species.
[0088] In other aspects of the invention, high agitation and
vigorous mixing is conducted during the addition of the linker-drug
moiety (AcBut-CM), for example, as achieved in part by addition of
the linker-drug moiety into the middle portion of the mixing
vortex, which is helpful in achieving low amounts of unconjugated
antibody, which is an improvement over prior methods.
[0089] In other aspects of the invention, the incubation time of
the reaction may be reduced to 2-5 minutes, compared to 60-90
minutes, to provide low aggregates and increase stability of
anti-EFNA4 ADCs. In another aspect of the invention, the amount of
ethanol (EtOH) in the reaction mixture may be reduced to 6-8%,
compared to 9%, to provide low aggregates and increase stability of
anti-EFNA4 ADCs.
[0090] In particular aspects of the invention, during purification,
the elution gradient may be optimized to provide a narrow DAR
distribution for anti-EFNA4 ADCs.
[0091] In particular aspects of the invention, purified anti-EFNA4
ADCs may have no unconjugated antibodies (free antibodies) present,
see FIG. 6. In other aspects of the invention, the purified
anti-EFNA4 ADCs may be monomeric ADCs, and the aggregates and
dimers are absent, see FIG. 7. In other aspects of the invention,
the purified anti-EFNA4 ADCs may have no free drug present, see
FIG. 8. In further aspects of the invention, the purified
anti-EFNA4 ADCs may be monomeric ADCs and have no free drug
present.
[0092] IIA. EFNA4 Antibodies
[0093] For preparation of EFNA4 antibody-drug conjugates of the
invention, the antibody, or antigen-binding fragment thereof, can
be any antibody, or antigen-binding fragment thereof, that
specifically binds to EFNA4. The antibodies the present invention
are further disclosed and characterized in PCT International
Publication No. WO2012/118547, which is incorporated herein by
reference in its entirety. The antibody, or antigen-binding
fragment thereof, may be isolated, purified, or derivatized for use
in preparation of EFNA antibody-drug conjugates.
[0094] An "antibody" or "Ab" is an immunoglobulin molecule capable
of recognizing and binding to a specific target or antigen, such as
a carbohydrate, polynucleotide, lipid, polypeptide, etc., through
at least one antigen recognition site, located in the variable
region of the immunoglobulin molecule. As used herein, the term
"antibody" can encompass any type of antibody, including but not
limited to monoclonal antibodies, polyclonal antibodies,
"antigen-binding fragments" (or portion), such as Fab, Fab',
F(ab').sub.2, Fd, Fv, Fc, etc., of intact antibodies that retain
the ability to specifically bind to a given antigen (e.g. EFNA4),
an isolated complementarity determining region (CDR), bispecific
antibodies, heteroconjugate antibodies, mutants thereof, fusion
proteins having an antibody, or antigen-binding fragment thereof,
(e.g., a domain antibody), single chain (ScFv) and single domain
antibodies (e.g., shark and camelid antibodies), maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR
and bis-scFv (see, e.g., Holliger and Hudson, 2005, Nature
Biotechnology 23(9): 1126-1136), humanized antibodies, chimeric
antibodies and any other modified configuration of the
immunoglobulin molecule that includes an antigen recognition site
of the required specificity, including glycosylation variants of
antibodies, amino acid sequence variants of antibodies, and
covalently modified antibodies. The antibodies may be murine, rat,
human, or any other origin (including chimeric or humanized
antibodies). In some aspects of the invention, the antibody, or
antigen-binding fragment thereof, of the disclosed EFNA4
antibody-drug conjugates is a chimeric, humanized, or a recombinant
human antibody, or EFNA4-binding fragment thereof.
[0095] An antibody, an antibody-drug conjugate, or a polypeptide
that "specifically binds" or "preferentially binds" (used
interchangeably herein) to a target or antigen (e.g., EFNA4
protein) is a term well understood in the art, and methods to
determine such specific or preferential binding are also well known
in the art. A molecule is said to exhibit "specific binding" or
"preferential binding" if it reacts or associates more frequently,
more rapidly, with greater duration and/or with greater affinity
with a particular cell or substance than it does with alternative
cells or substances. An antibody "specifically binds" or
"preferentially binds" to a target or antigen if it binds with
greater affinity, avidity, more readily, and/or with greater
duration than it binds to other substances. For example, an
antibody that specifically or preferentially binds to an EFNA4
epitope is an antibody that binds this epitope with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other EFNA4 epitopes or non-EFNA4 epitopes.
[0096] The term "binding affinity" or "K.sub.D" as used herein, is
intended to refer to the equilibrium dissociation constant of a
particular antigen-antibody interaction. The K.sub.D is the ratio
of the rate of dissociation, also called the "off-rate" or
"k.sub.d", to the rate of association, or "on-rate" or "k.sub.a".
Thus, K.sub.D equals k.sub.d/k.sub.a and is expressed as a molar
concentration (M). It follows that the smaller the K.sub.D, the
stronger the binding affinity. Therefore, a K.sub.D of 1 .mu.M
indicates weak binding affinity compared to a K.sub.D of 1 nM.
K.sub.D values for antibodies can be determined using methods well
established in the art. One method for determining the K.sub.D of
an antibody is by using surface plasmon resonance, typically using
a biosensor system such as a Biacore.RTM. system.
[0097] Specific binding of the disclosed EFNA4 antibody-drug
conjugates refers to a preferential binding of an antibody to human
EFNA4 antigen in a heterogeneous sample having multiple different
antigens. Typically, specific binding occurs if the binding
affinity is at least about 10.sup.-7 M or higher, such as at least
about 10.sup.-8 M or higher, including at least about 10.sup.-9 M
or higher, at least about 10.sup.-11 M or higher, or at least about
10.sup.-12 M or higher. For example, specific binding of an
antibody of the invention to a human EFNA4 antigen includes binding
in the range of at least about 1.times.10.sup.-7 M to about
1.times.10.sup.-12 M, such as within the range of about
1.times.10.sup.-8 M to about 1.times.10.sup.-12 M, or within the
range of about 1.times.10.sup.-8 M to about 1.times.10.sup.-11 M,
or within the range of about 1.times.10.sup.-8 M to about
1.times.10.sup.-10 M, or within the range of about1.times.10.sup.-9
M to about 1.times.10.sup.-10 M. Specific binding also refers to
selective targeting of an EFNA4 antibody, or antigen-binding
fragment thereof, to EFNA4-expressing cells following
administration of the antibody to a subject.
[0098] As used herein, "epitope" includes any protein determinant
capable of specific binding to an immunoglobulin or T-cell receptor
or otherwise interacting with a molecule. Epitopic determinants
generally consist of chemically active surface groupings of
molecules such as amino acids or carbohydrate or sugar side chains
and generally have specific three dimensional structural
characteristics, as well as specific charge characteristics. An
epitope may be `linear` or `conformational.` In a linear epitope,
all of the points of interaction between the protein and the
interacting molecule (such as an antibody) occur linearally along
the primary amino acid sequence of the protein. In a conformational
epitope, the points of interaction occur across amino acid residues
on the protein that are separated from one another. Once a desired
epitope on an antigen is determined, it is possible to generate
antibodies to that epitope, e.g., using the techniques described in
the present invention. Alternatively, during the discovery process,
the generation and characterization of antibodies may elucidate
information about desirable epitopes. From this information, it is
then possible to competitively screen antibodies for binding to the
same epitope. An approach to achieve this is to conduct
cross-competition studies to find antibodies that competitively
bind with one another, i.e. the antibodies compete for binding to
the antigen. A high throughput process for `binning` antibodies
based upon their cross-competition is described in PCT
International Publication No. WO 03/48731. As used herein, the term
`binning` refers to a method to group antibodies based on their
antigen binding characteristics and competition with each
other.
[0099] An isolated antibody that specifically binds EFNA4 may,
however, have cross-reactivity to other antigens, such as EFNA4
molecules from other species (i.e. an ortholog) or with more than
one isoform of EFNA4. An "isolated antibody", as used herein,
refers to an antibody that is substantially free of other
antibodies having different antigenic specificities (e.g., an
isolated antibody that specifically binds EFNA4 is substantially
free of antibodies that specifically bind antigens other than
EFNA4). Moreover, an isolated antibody may be substantially free of
other cellular material and/or chemicals. It is also understood
that by reading this definition, for example, an antibody (or
moiety or epitope) that specifically or preferentially binds to a
first target may or may not specifically or preferentially bind to
a second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0100] In some aspects of the invention, an EFNA4 antibody-drug
conjugate includes an antibody that competes for binding to human
EFNA4 with, and/or binds the same epitope as, an antibody, or
antigen-binding fragment thereof, having (a) a heavy chain variable
region set forth as SEQ ID NO: 13 and a light chain variable region
set forth as SEQ ID NO: 27; or (b) a heavy chain variable region
set forth as SEQ ID NO: 39 and a light chain variable region set
forth as SEQ ID NO: 53.
[0101] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
fragment thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
fragment thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0102] Native or naturally occurring antibodies, and native
immunoglobulins are typically heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light (L) chains
and two identical heavy (H) chains. Each light chain is linked to a
heavy chain by one covalent disulfide bond, while the number of
disulfide linkages varies among the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (VH) followed by a number of constant
domains. Each light chain has a variable domain at one end (VL) and
a constant domain at its other end; the constant domain of the
light chain is aligned with the first constant domain of the heavy
chain, and the light chain variable domain is aligned with the
variable domain of the heavy chain. Particular amino acid residues
are believed to form an interface between the light- and
heavy-chain variable domains. The term "variable" refers to the
fact that certain portions of the variable domains differ
extensively in sequence among antibodies.
[0103] Antibodies and the above-noted antibody domains may be
described as "polypeptides", "oligopeptides", "peptides" and
"proteins", i.e., chains of amino acids of any length, preferably,
relatively short (e.g., 10-100 amino acids). The chain may be
linear or branched, it may comprise modified amino acids, and/or
may be interrupted by non-amino acids. The terms also encompass an
amino acid chain that has been modified naturally or by
intervention; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other manipulation
or modification, such as conjugation with a labeling component.
Also included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), as well as other
modifications known in the art. It is understood that the
polypeptides can occur as single chains or associated chains. Amino
acids may be referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical.
[0104] A "constant region" of an antibody refers to the constant
region of the antibody light chain or the constant region of the
antibody heavy chain, either alone or in combination. The constant
regions of chimeric and humanized EFNA4 antibodies may be derived
from constant regions of any one of IgA, IgD, IgE, IgG, IgM, any
isotypes thereof (e.g., IgG1, IgG2, IgG3, or IgG4 isotypes of IgG),
as well as subclasses and mutated versions thereof. Depending on
the antibody amino acid sequence of the constant region of its
heavy chains, immunoglobulins can be assigned to different classes.
There are five major classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The heavy chain constant regions that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0105] The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effector functions,
such as Fc receptor (FcR) binding, participation of the antibody in
antibody-dependent cellular toxicity, opsonization, initiation of
complement dependent cytotoxicity, and mast cell degranulation. As
known in the art, the term "Fc region" is used to define a
C-terminal region of an immunoglobulin heavy chain. The "Fc region"
may be a native sequence Fc region or a variant Fc region. Although
the boundaries of the Fc region of an immunoglobulin heavy chain
might vary, the human IgG heavy chain Fc region is usually defined
to stretch from an amino acid residue at position Cys226, or from
Pro230, to the carboxyl-terminus thereof. The numbering of the
residues in the Fc region is that of the EU index as in Kabat.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally having two
constant regions, CH2 and CH3.
[0106] As used in the art, "Fc receptor" and "FcR" describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991;
Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J.
Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol., 117:587-593 (1976);
and Kim et al., European J. Immunol., 24:2429-2434 (1994)).
[0107] In some aspects of the invention, the antibody, or
antigen-binding fragment thereof, of the disclosed EFNA4
antibody-drug conjugates includes an IgG1 heavy chain constant
region, for example a huE22 heavy chain set forth as SEQ ID NO: 25
or a huE47 heavy chain set forth as SEQ ID NO: 51. In other
aspects, the antibody, or antigen-binding fragment thereof, of the
disclosed EFNA4 antibody-drug conjugates includes a kappa light
chain constant region, for example a huE22 light chain set forth as
SEQ ID NO: 37 or a huE47 light chain set forth as SEQ ID NO: 63. In
particular aspects of the invention, an EFNA4 antibody-drug
conjugate can include an IgG1 heavy chain constant region and a
kappa light chain constant region, for example, a heavy chain set
forth as SEQ ID NO: 25 and a light chain set forth as SEQ ID NO:
37, or as another example, a heavy chain set forth as SEQ ID NO: 51
and a light chain set forth as SEQ ID NO: 63.
[0108] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FR) connected by three
complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen binding site of
antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948
(1997),). As used herein, a CDR may refer to CDRs defined by either
approach or by a combination of both approaches.
[0109] A CDR of a variable domain are amino acid residues within
the variable region that are identified in accordance with the
definitions of the Kabat, Chothia, the accumulation of both Kabat
and Chothia, VBASE2, AbM, contact, and/or conformational
definitions or any method of CDR determination well known in the
art. Antibody CDRs may be identified as the hypervariable regions
originally defined by Kabat et al. See, e.g., Kabat et al., 1992,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, NIH, Washington D.C. The positions of the CDRs may
also be identified as the structural loop structures originally
described by Chothia and others. See, e.g., Chothia et al., Nature
342:877-883, (1989). The CDR positions may also be derived from an
analysis of the VBASE2 database. (See, e.g. Retter et al., Nucleic
Acids Res. 33(Database Issue):D671-D674, 2005 and
http://www.vbase2.org/).
[0110] Other approaches to CDR identification include the "AbM
definition," which is a compromise between Kabat and Chothia and is
derived using Oxford Molecular's AbM antibody modeling software
(now Accelrys.RTM.), or the "contact definition" of CDRs based on
observed antigen contacts, set forth in MacCallum et al., J. Mol.
Biol., 262:732-745, (1996). In another approach, referred to herein
as the "conformational definition" of CDRs, the positions of the
CDRs may be identified as the residues that make enthalpic
contributions to antigen binding. See, e.g., Makabe et al., Journal
of Biological Chemistry, 283:1156-1166, 2008. Still other CDR
boundary definitions may not strictly follow one of the above
approaches, but will nonetheless overlap with at least a portion of
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. As used herein, a CDR may
refer to CDRs defined by any approach known in the art, including
combinations of approaches. The methods used herein may utilize
CDRs defined according to any of these approaches. For EFNA4
antibody-drug conjugates described herein, CDRs may be defined in
accordance with any of Kabat, Chothia, extended, VBASE2, AbM,
contact, and/or conformational definitions.
[0111] In other aspects of the invention, the EFNA4 antibody, or
antigen-binding fragment thereof, includes one or more CDR(s) of
the antibody (such as one, two, three, four, five, or all six
CDRs).
[0112] For the instant invention, the CDRs set forth in Table 2
below (SEQ ID NOS: 5-64) were derived using Kabat and Chothia
approaches and the CDRS set forth in FIG. 2 were derived from an
analysis of the VBASE2 database. Accordingly, antibodies having
CDRs defined by all such nomenclature are expressly included within
the scope of the instant invention. More broadly, the term
"variable region CDR amino acid residue" includes amino acids in a
CDR as identified using any sequence or structure based method as
set forth above.
[0113] In some aspects of the invention, an EFNA4 antibody-drug
conjugate includes an antibody, or antigen-binding fragment
thereof, having CDRs of a huE22 antibody. For example, an EFNA4
antibody-drug conjugate may include an antibody, or antigen-binding
fragment thereof, including at least one heavy chain variable
region and at least one light chain variable region, wherein the at
least one heavy chain variable region has three CDRs set forth as
SEQ ID NOs: 15, 19, and 23. In some aspects of the invention, an
EFNA4 antibody-drug conjugate includes an antibody, or
antigen-binding fragment thereof, having at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one light chain variable region has three CDRs
set forth as SEQ ID NOs: 29, 33, and 35. An EFNA4 antibody-drug
conjugate of the invention can also include an antibody, or
antigen-binding fragment thereof, including (a) a heavy chain
variable region having three CDRs set forth as SEQ ID NOs: 15, 19,
and 23; and (b) a light chain variable region having three CDRs set
forth as SEQ ID NOs: 29, 33, and 35.
[0114] In still other aspects of the invention, an EFNA4
antibody-drug conjugate includes an antibody, or antigen-binding
fragment thereof, having one or more huE22 CDRs defined according
to Chothia or derived from an analysis of the VBASE2 database. For
example, an EFNA4 antibody-drug conjugate can include an antibody,
or antigen-binding fragment thereof, having at least one heavy
chain variable region and at least one light chain variable region,
wherein the at least one heavy chain variable region includes three
huE22 CDRs defined by Chothia (see Table 2) or three huE22 CDRs
derived from an analysis of the VBASE2 database (see FIG. 2). As
another example, an EFNA4 antibody-drug conjugate can include an
antibody, or antigen-binding fragment thereof, having at least one
heavy chain variable region and at least one light chain variable
region, wherein the at least one light chain variable region
includes three huE22 CDRs defined by Chothia (see Table 2) or three
huE22 CDRs derived from an analysis of the VBASE2 database (see
FIG. 2). In some aspects of the invention, an EFNA4 antibody-drug
conjugate of the invention can include an antibody, or
antigen-binding fragment thereof, having (a) a heavy chain variable
region having three huE22 CDRs defined according to Chothia (see
Table 2); and (b) a light chain variable region having three huE22
CDRs defined according to Chothia (see Table 2). In some aspects of
the invention, an EFNA4 antibody-drug conjugate of the invention
can include an antibody, or antigen-binding fragment thereof,
having (a) a heavy chain variable region including three huE22 CDRs
derived from an analysis of the VBASE2 database (see FIG. 2); and
(b) a light chain variable region including three huE22 CDRs
derived from an analysis of the VBASE2 database (see FIG. 2).
[0115] In other aspects of the invention, an EFNA4 antibody-drug
conjugate includes an antibody, or antigen-binding fragment
thereof, having CDRs of a huE47 antibody. For example, an EFNA4
antibody-drug conjugate may include an antibody, or antigen-binding
fragment thereof, having at least one heavy chain variable region
and at least one light chain variable region, wherein the at least
one heavy chain variable region includes three CDRs set forth as
SEQ ID NOs: 41, 45, and 49. In some aspects of the invention, an
EFNA4 antibody-drug conjugate includes an antibody, or
antigen-binding fragment thereof, having at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one light chain variable region includes three
CDRs set forth as SEQ ID NOs: 55, 59, and 61. An EFNA4
antibody-drug conjugate of the invention can also include (a) a
heavy chain variable region having three CDRs set forth as SEQ ID
NOs: 41, 45, and 49; and (b) a light chain variable region having
three CDRs set forth as SEQ ID NOs: 55, 59, and 61.
[0116] In still other aspects of the invention, an EFNA4
antibody-drug conjugate includes an antibody, or antigen-binding
fragment thereof, having one or more huE47 CDRs defined according
to Chothia or derived from an analysis of the VBASE2 database. For
example, an EFNA4 antibody-drug conjugate can include an antibody,
or antigen-binding fragment thereof, having at least one heavy
chain variable region and at least one light chain variable region,
wherein the at least one heavy chain variable region includes three
huE47 CDRs defined by Chothia (see Table 2) or three huE47 CDRs
derived from an analysis of the VBASE2 database (see FIG. 2). As
another example, an EFNA4 antibody-drug conjugate can include an
antibody, or antigen-binding fragment thereof, having at least one
heavy chain variable region and at least one light chain variable
region, wherein the at least one light chain variable region
includes three huE47 CDRs defined by Chothia (see Table 2) or three
huE47 CDRs derived from an analysis of the VBASE2 database (see
FIG. 2). In some aspects of the invention, an EFNA4 antibody-drug
conjugate of the invention can include an antibody, or
antigen-binding fragment thereof, having (a) a heavy chain variable
region including three huE47 CDRs defined according to Chothia (see
Table 2); and (b) a light chain variable region including three
huE47 CDRs defined according to Chothia (see Table 2). In some
aspects of the invention, an EFNA4 antibody-drug conjugate of the
invention can include an antibody, or antigen-binding fragment
thereof, having (a) a heavy chain variable region having three
huE47 CDRs derived from an analysis of the VBASE2 database (see
FIG. 2); and (b) a light chain variable region having three huE47
CDRs derived from an analysis of the VBASE2 database (see FIG.
2).
[0117] In some aspects of the invention, antibodies used to prepare
EFNA4 antibody-drug conjugates of the invention will be monoclonal
antibodies. The term "monoclonal antibody" or "mAb" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations, which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler and
Milstein, Nature 256:495-497, 1975, or may be made by recombinant
DNA methods such as described in U.S. Pat. No. 4,816,567. The
monoclonal antibodies may also be isolated from phage libraries
generated using the techniques described in McCafferty et al.,
Nature 348:552-554, 1990, for example.
[0118] In some aspects of the invention, antibodies used to prepare
antibody-drug conjugates of the invention will be monovalent, i.e.,
having one antigen binding site per molecule (e.g., IgG or Fab). In
some instances, a monovalent antibody can have more than one
antigen binding sites, but the binding sites are from different
antigens. In some aspects of the invention, the antibody, or
antigen-binding fragment thereof, of an antibody-drug conjugate of
the invention may include a "bivalent antibody", i.e., having two
antigen binding sites per molecule (e.g., IgG). In some instances,
the two binding sites have the same antigen specificities.
Alternatively, bivalent antibodies may be bispecific. A
"bispecific," "dual-specific" or "bifunctional" antibody is a
hybrid antibody having two different antigen binding sites. The two
antigen binding sites of a bispecific antibody bind to two
different epitopes, which may reside on the same or different
protein targets.
[0119] The term "chimeric antibody" is intended to refer to
antibodies in which part or all of the variable region sequences
are derived from one species and the constant region sequences are
derived from another species, such as an antibody in which the
variable region sequences are derived from a mouse antibody and the
constant region sequences are derived from a human antibody.
[0120] As used herein, "humanized" or "CDR grafted" antibody refers
to forms of non-human (e.g. murine) antibodies that are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen binding
subsequences of antibodies) that contain minimal sequence derived
from a non-human immunoglobulin. Preferably, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from one or more complementary determining regions (CDRs) of the
recipient are replaced by residues from one or more CDRs of a
non-human species (donor antibody) such as mouse, rat, or rabbit
having the desired specificity, affinity, and capacity.
[0121] In some instances, Fv framework region (FR) residues of the
human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, the humanized antibody may include residues
that are found neither in the recipient antibody nor in the
imported CDR or framework sequences, but are included to further
refine and optimize antibody performance. In general, the humanized
antibody will include substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will include at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. In some aspects of the invention the
antibodies have Fc regions modified as described in PCT
International Publication No. WO 99/58572. Other forms of humanized
antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1,
CDR H2, or CDR H3) which may be altered with respect to the
original antibody, which are also termed one or more CDRs "derived
from" one or more CDRs from the original antibody.
[0122] Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al. Nature 321:522-525
(1986); Riechmann et al. Nature 332:323-327 (1988); Verhoeyen et
al. Science 239:1534-1536 (1988)), by substituting rodent or mutant
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089;
5,693,761; 5,693,762; 5,859,205; which are incorporated herein by
reference in its entirety. In some instances, residues within the
framework regions of one or more variable regions of the human
immunoglobulin are replaced by corresponding non-human residues
(see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762;
and 6,180,370). Furthermore, humanized antibodies may include
residues that are not found in the recipient antibody or in the
donor antibody. These modifications are made to further refine
antibody performance (e.g., to obtain desired affinity). In
general, the humanized antibody will include substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
framework regions are those of a human immunoglobulin sequence. The
humanized antibody optionally also will include at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. For further details see Jones et al. Nature
321:522-525 (1986); Riechmann et al. Nature 332:323-327(1988); and
Presta Curr. Op. Struct. Biol. 2:593-596 (1992); which are
incorporated herein by reference in its entirety. Accordingly, such
"humanized" antibodies may include antibodies wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some framework residues are substituted
by residues from analogous sites in rodent antibodies. See, for
example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762;
5,859,205. See also U.S. Pat. No. 6,180,370, and PCT International
Publication No. WO 01/27160, where humanized antibodies and
techniques for producing humanized antibodies having improved
affinity for a predetermined antigen are disclosed.
[0123] "Recombinant human antibody" or "fully human antibody"
refers to those antibodies having an amino acid sequence
corresponding to that of an antibody produced by a human and/or
which has been made using any of the techniques for making human
antibodies known to those skilled in the art or disclosed herein.
This definition of a human antibody includes antibodies having at
least one human heavy chain polypeptide or at least one human light
chain polypeptide. One such example is an antibody having murine
light chain and human heavy chain polypeptides. Human antibodies
can be produced using various techniques known in the art. For
example, a human antibody is selected from a phage library, where
that phage library expresses human antibodies (Vaughan et al.,
Nature Biotechnology, 14:309-314, (1996); Sheets et al., Proc.
Natl. Acad. Sci. (USA) 95:6157-6162, (1998); Hoogenboom and Winter,
J. Mol. Biol., 227:381-388, (1992); Marks et al., J. Mol. Biol.,
222:581-597, (1991)). Human antibodies can also be made by
immunization of animals into which human immunoglobulin loci have
been transgenically introduced in place of the endogenous loci,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. This approach is described in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; and 5,661,016. Alternatively, the human antibody may be
prepared by immortalizing human B lymphocytes that produce an
antibody directed against a target antigen (such B lymphocytes may
be recovered from an individual or from single cell cloning of the
cDNA, or may have been immunized in vitro). See, e.g., Cole et al.
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77,
(1985); Boerner et al., J. Immunol., 147 (1):86-95, (1991); and
U.S. Pat. No. 5,750,373.
[0124] Antibodies of the invention can be produced using techniques
well known in the art, e.g., recombinant technologies, phage
display technologies, synthetic technologies or combinations of
such technologies or other technologies readily known in the art
(see, for example, Jayasena, S. D., Clin. Chem., 45: 1628-50 (1999)
and Fellouse, F. A., et al, J. Mol. Biol., 373(4):924-40 (2007)).
Additional guidance may be found in Sambrook J. & Russell D.
Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al.,
Short Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, Wiley, John & Sons,
Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1998); and Coligan et al., Short Protocols in Protein Science,
Wiley, John & Sons, Inc. (2003). Representative methods are
also described in Examples 1-3 herein below.
[0125] In general, for the production of hybridoma cell lines, the
route and schedule of immunization of the host animal are generally
in keeping with established and conventional techniques for
antibody stimulation and production. It is contemplated that any
mammalian subject including humans or antibody producing cells
therefrom can be manipulated to serve as the basis for production
of mammalian, including human and hybridoma cell lines. Typically,
the host animal is inoculated intraperitoneally, intramuscularly,
orally, subcutaneously, intraplantar, and/or intradermally with an
amount of immunogen, including as described herein.
[0126] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C., Nature
256:495-497, 1975 or as modified by Buck, D. W., et al., In Vitro,
18:377-381, 1982. Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the EFNA4 monoclonal antibodies of the
subject invention. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay). Hybridomas that
may be used as source of antibodies encompass all derivatives,
progeny cells of the parent hybridomas that produce monoclonal
antibodies specific for EFNA4, or a portion thereof.
[0127] Hybridomas that produce such antibodies may be grown in
vitro or in vivo using known procedures. The monoclonal antibodies
may be isolated from the culture media or body fluids, by
conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired
activity, if present, can be removed, for example, by running the
preparation over adsorbents made of the immunogen attached to a
solid phase and eluting or releasing the desired antibodies off the
immunogen. Immunization of a host animal with a human EFNA4, or a
fragment containing the target amino acid sequence conjugated to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups, can yield a population of antibodies (e.g.,
monoclonal antibodies).
[0128] If desired, the EFNA4 antibody (monoclonal or polyclonal) of
interest may be sequenced and the polynucleotide sequence may then
be cloned into a vector for expression or propagation. The sequence
encoding the antibody of interest may be maintained in vector in a
host cell and the host cell can then be expanded and frozen for
future use. Production of recombinant monoclonal antibodies in cell
culture can be carried out through cloning of antibody genes from B
cells by means known in the art. See, e.g. Tiller et al., J.
Immunol. Methods 329:112-124, 2008; U.S. Pat. No. 7,314,622.
[0129] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0130] The term "vector" means a construct, which is capable of
delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0131] The term "expression control sequence" means a nucleic acid
sequence that directs transcription of a nucleic acid. An
expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0132] Alternatively, the polynucleotide sequence may be used for
genetic manipulation to "humanize" the antibody or to improve the
affinity, or other characteristics of the antibody. For example,
the constant region may be engineered to more nearly resemble human
constant regions to avoid immune response if the antibody is used
in clinical trials and treatments in humans. It may be desirable to
genetically manipulate the antibody sequence to obtain greater
affinity to EFNA4 and greater efficacy in inhibiting EFNA4.
[0133] There are four general steps that may be used to humanize a
monoclonal antibody: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process (3) the actual humanizing
methodologies/techniques and (4) the transfection and expression of
the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; and 6,180,370.
[0134] Humanized antibodies may be prepared using any one of a
variety of methods including veneering, grafting of complementarity
determining regions (CDRs), grafting of abbreviated CDRs, grafting
of specificity determining regions (SDRs), and Frankenstein
assembly, as described below. Humanized antibodies also include
superhumanized antibodies, in which one or more changes have been
introduced in the CDRs. For example, human residues may be
substituted for non-human residues in the CDRs. These general
approaches may be combined with standard mutagenesis and synthesis
techniques to produce an anti-EFNA4 antibody of any desired
sequence.
[0135] Veneering is based on the concept of reducing potentially
immunogenic amino acid sequences in a rodent or other non-human
antibody by resurfacing the solvent accessible exterior of the
antibody with human amino acid sequences. Thus, veneered antibodies
appear less foreign to human cells than the unmodified non-human
antibody. See Padlan (1991) Mol. Immunol. 28:489-98. A non-human
antibody is veneered by identifying exposed exterior framework
region residues in the non-human antibody, which are different from
those at the same positions in framework regions of a human
antibody, and replacement of the identified residues with amino
acids that typically occupy these same positions in human
antibodies.
[0136] Grafting of CDRs is performed by replacing one or more CDRs
of an acceptor antibody (e.g., a human antibody or other antibody
having desired framework residues) with CDRs of a donor antibody
(e.g., a non-human antibody). Acceptor antibodies may be selected
based on similarity of framework residues between a candidate
acceptor antibody and a donor antibody. For example, according to
the Frankenstein approach, human framework regions are identified
as having substantial sequence homology to each framework region of
the relevant non-human antibody, and CDRs of the non-human antibody
are grafted onto the composite of the different human framework
regions. A related method also useful for preparation of antibodies
of the invention is described in U.S. Patent Application
Publication No. 2003/0040606.
[0137] Grafting of abbreviated CDRs is a related approach.
Abbreviated CDRs include the specificity-determining residues and
adjacent amino acids, including those at positions 27d-34, 50-55
and 89-96 in the light chain, and at positions 31-35b, 50-58, and
95-101 in the heavy chain (numbering convention of (Kabat et al.
(1987)). See (Padlan et al. (1995) FASEB J. 9: 133-9). Grafting of
specificity-determining residues (SDRs) is premised on the
understanding that the binding specificity and affinity of an
antibody combining site is determined by the most highly variable
residues within each of the complementarity determining regions
(CDRs). Analysis of the three-dimensional structures of
antibody-antigen complexes, combined with analysis of the available
amino acid sequence data may be used to model sequence variability
based on structural dissimilarity of amino acid residues that occur
at each position within the CDR. SDRs are identified as minimally
immunogenic polypeptide sequences consisting of contact residues.
See Padlan et al. (1995) FASEB J. 9: 133-139.
[0138] In general, human acceptor frameworks are selected on the
basis that they are substantially similar to the framework regions
of the donor antibodies, or which are most similar to the consensus
sequence of the variable region subfamily. Following grafting,
additional changes may be made in the donor and/or acceptor
sequences to optimize antibody binding, functionality, codon usage,
expression levels, etc, including introduction of non-human
residues into the framework regions. See e.g., PCT International
Publication No. WO 91/09967.
[0139] For grafting of CDRs onto a heavy chain variable framework
region, useful framework sequences may be derived from a DP-21
(VH7), DP-54 (VH3-07), DP-47 (VH3-23), DP-53 (VH-74), DP-49
(VH3-30), DP-48 (VH3-13), DP-75, DP-8(VH1-2), DP-25, VI-2b and VI-3
(VH1-03), DP-15 and V1-8 (VH1-08), DP-14 and V1-18 (VH1-18), DP-5
and V1-24P (VH1-24), DP-4 (VH1-45), DP-7 (VH1-46), DP-10, DA-6 and
YAC-7 (VH1-69), DP-88 (VH1-e), DP-3 and DA-8 (VH1-f). For grafting
of CDRs onto a light chain variable framework region, useful
framework sequences may be derived from a DPK24 subgroup IV germ
line clone, a Will subgroup (DPK23, DPK22, DPK20, DPK21), or a
V.kappa.I subgroup germ line clone (DPK9, DPK1, O2, DPK7).
[0140] Antigen-binding fragments or antibody fragments can be
produced by proteolytic or other degradation of the antibodies, by
recombinant methods (i.e., single or fusion polypeptides) as
described above or by chemical synthesis. Polypeptides of the
antibodies, especially shorter polypeptides up to about 50 amino
acids, are conveniently made by chemical synthesis. Methods of
chemical synthesis are known in the art and are commercially
available. For example, an antibody or antibody fragment could be
produced by an automated polypeptide synthesizer employing the
solid phase method. See also, U.S. Pat. Nos. 5,807,715; 4,816,567;
and 6,331,415.
[0141] In other aspects of the invention, the EFNA4 antibody-drug
conjugates include an antibody, or antigen-binding fragment
thereof, having a huE5, huE15, huE22, or huE47 heavy chain and/or
light chain variable region, or a variable region substantially
similar to a huE5, huE15, huE22, or huE47 heavy chain or light
chain variable region.
[0142] As applied to polypeptides, the term "substantial identity"
or "substantial similarity" means that two amino acid sequences,
when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights as supplied with the programs, share at
least 70%, 75% or 80% sequence identity, preferably at least 90% or
95% sequence identity, and more preferably at least 97%, 98% or 99%
sequence identity. In some substantially similar amino acid
sequences, residue positions that are not identical differ by
conservative amino acid substitutions.
[0143] Substantially similar polypeptides also include
conservatively substituted variants in which one or more residues
have been conservatively substituted with a functionally similar
residue. Examples of conservative substitutions include the
substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another; the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine; the substitution of one basic residue
such as lysine, arginine or histidine for another; or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another.
[0144] A further indication that two proteins are substantially
identical is that they share an overall three-dimensional
structure, or are biologically functional equivalents.
[0145] In some aspects of the invention, an antibody-drug
conjugate, which binds to EFNA4, including an antibody, or
antigen-binding fragment thereof, having a heavy chain variable
region set forth as any one of SEQ ID NOs: 5, SEQ ID NO: 9, SEQ ID
NO: 13 and SEQ ID NO: 39 and/or a light chain variable region set
forth as any one of SEQ ID NOs: 7, SEQ ID NO: 11, SEQ ID NO: 27 and
SEQ ID NO: 53. For example, an EFNA4 antibody-drug conjugate of the
invention can include an antibody, or antigen-binding fragment
thereof, having a heavy chain variable region having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 13 and a
light chain variable region having an amino acid sequence that is
at least 90% identical to SEQ ID NO: 27; or an antibody, or
antigen-binding fragment thereof, having a heavy chain variable
region set forth as SEQ ID NO: 13 and a light chain variable region
having an amino acid sequence set forth as SEQ ID NO: 27. As
another example, an EFNA4 antibody-drug conjugate of the invention
can include an antibody, or antigen-binding fragment thereof,
having a heavy chain variable region having an amino acid sequence
that is at least 90% identical to SEQ ID NO: 39 and a light chain
variable region having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 53; or an antibody, or antigen-binding
fragment thereof, having a heavy chain variable region set forth as
SEQ ID NO: 39 and a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 53.
[0146] To express the EFNA4 antibodies of the present invention,
DNA fragments encoding VH and VL regions can first be obtained
using any of the methods described above. As known in the art,
"polynucleotide," "nucleic acid/nucleotide," and "oligonucleotide"
are used interchangeably herein, and include polymeric forms of
nucleotides of any length, either deoxyribonucleotides or
ribonucleotides, analogs thereof, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. Polynucleotides
may have any three-dimensional structure, and may perform any
function, known or unknown. The following are non-limiting examples
of polynucleotides: a gene or gene fragment, exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, DNA,
cDNA, genomic DNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers.
Polynucleotides may be naturally-occurring, synthetic, recombinant
or any combination thereof. A polynucleotide may include modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the chain. The sequence of nucleotides
may be interrupted by non-nucleotide components. A polynucleotide
may be further modified after polymerization, such as by
conjugation with a labeling component. Other types of modifications
include, for example, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as, for example, those with uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoamidates,
carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, poly-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as unmodified forms of the polynucleotide(s). Further, any of
the hydroxyl groups ordinarily present in the sugars may be
replaced, for example, by phosphonate groups, phosphate groups,
protected by standard protecting groups, or activated to prepare
additional linkages to additional nucleotides, or may be conjugated
to solid supports. The 5' and 3' terminal OH can be phosphorylated
or substituted with amines or organic capping group moieties of
from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized
to standard protecting groups. Polynucleotides can also contain
analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or
beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic
analogs and abasic nucleoside analogs such as methyl riboside. One
or more phosphodiester linkages may be replaced by alternative
linking groups. These alternative linking groups include, but are
not limited to, features wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0147] Representative DNAs encoding anti-EFNA4 antibody heavy chain
and light chain variable regions are set forth as SEQ ID NOs: 6
(huE5 VH DNA), 8 (huE5 VL DNA), 10 (huE15 VH DNA), 12 (huE15 VL
DNA), 14 (huE22 VH DNA), 28 (huE22 VL DNA), 40 (huE47 VH DNA), and
54 (huE47 VL DNA). Representative DNAs encoding anti-EFNA antibody
heavy chains and light chains are set forth as SEQ ID NO: 26 (huE22
HC), SEQ ID NO: 28 (huE22 LC), SEQ ID NO: 52 (huE47 HC), and SEQ ID
NO: 64 (huE47 LC). See Table 2 herein below. The CDRs of the huE5
and huE15 antibodies are indicated by underlining in Table 2. The
CDRs of the huE22 and huE47 antibodies are set forth as separate
sequences and sequence identifiers in Table 2 (defined by Kabat or
Chothia) or in FIG. 2 (derived from an analysis of the VBASE2
database).
[0148] Various modifications, e.g. mutations, substitutions,
deletions, and/or additions can also be introduced into the huE5,
huE15, huE22, and huE47 DNA sequences using standard methods known
to those of skill in the art. For example, mutagenesis can be
carried out using standard methods, such as PCR-mediated
mutagenesis, in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis.
[0149] Accordingly, based upon the disclosure of the instant
application, one skilled in the art would readily recognize the
sequences of DNAs substantially similar huE5, huE15, huE22, and
huE47 DNAs. The term "substantial similarity" or "substantial
sequence similarity," when referring to a nucleic acid or fragment
thereof, means that when optimally aligned with appropriate
nucleotide insertions or deletions with another nucleic acid (or
its complementary strand), there is nucleotide sequence identity in
at least about 85%, preferably at least about 90%, and more
preferably at least about 95%, 96%, 97%, 98% or 99% of the
nucleotide bases, as measured by any well-known algorithm of
sequence identity, such as FASTA, BLAST or Gap.
[0150] The term "percent sequence identity" in the context of
nucleic acid sequences means the residues in two sequences that are
the same when aligned for maximum correspondence. The length of
sequence identity comparison may be over a stretch of at least
about nine nucleotides, usually at least about 18 nucleotides, more
usually at least about 24 nucleotides, typically at least about 28
nucleotides, more typically at least about 32 nucleotides, and
preferably at least about 36, 48 or more nucleotides. There are a
number of different algorithms known in the art which can be used
to measure nucleotide sequence identity. For instance,
polynucleotide sequences can be compared using FASTA, Gap or
Bestfit, which are programs in Wisconsin Package Version 10.0,
Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes,
e.g., the programs FASTA2 and FASTA3, provides alignments and
percent sequence identity of the regions of the best overlap
between the query and search sequences (Pearson, Methods Enzymol.
183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000);
Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol.
Biol. 276:71-84 (1998); which are incorporated herein by reference
in its entirety). Unless otherwise specified, default parameters
for a particular program or algorithm are used. For instance,
percent sequence identity between nucleic acid sequences can be
determined using FASTA with its default parameters (a word size of
6 and the NOPAM factor for the scoring matrix) or using Gap with
its default parameters as provided in GCG Version 6.1, which is
incorporated herein by reference in its entirety.
[0151] A further indication that two nucleic acid sequences are
substantially identical is that proteins encoded by the nucleic
acids are substantially identical, share an overall
three-dimensional structure, or are biologically functional
equivalents. These terms are defined further herein below. Nucleic
acid molecules that do not hybridize to each other under stringent
conditions are still substantially identical if the corresponding
proteins are substantially identical. This may occur, for example,
when two nucleotide sequences comprise conservatively substituted
variants as permitted by the genetic code.
[0152] Conservatively substituted variants are nucleic acid
sequences having degenerate codon substitutions wherein the third
position of one or more selected (or all) codons is substituted
with mixed-base and/or deoxyinosine residues. See Batzer et al.
(1991) Nucleic Acids Res. 19:5081; Ohtsuka et al. (1985) J. Biol.
Chem. 260:2605-2608; and Rossolini et al. (1994) Mol. Cell Probes
8:91-98.
[0153] One type of substitution, for example, that may be made is
to change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant region of an antibody. As another example, the cysteine
may be canonical.
[0154] The antibodies may also be modified, e.g. in the variable
domains of the heavy and/or light chains, e.g., to alter a binding
property of the antibody. For example, a mutation may be made in
one or more of the CDR regions to increase or decrease the K.sub.D
of the antibody for EFNA4, to increase or decrease k.sub.off, or to
alter the binding specificity of the antibody. Techniques in
site-directed mutagenesis are well-known in the art. See, e.g.,
Sambrook et al. and Ausubel et al., supra.
[0155] A modification or mutation may also be made in a framework
region or constant region to increase the half-life of an EFNA4
antibody. See, e.g. PCT International Publication No. WO 00/09560.
A mutation in a framework region or constant region can also be
made to alter the immunogenicity of the antibody, to provide a site
for covalent or non-covalent binding to another molecule, or to
alter such properties as complement fixation, FcR binding and
antibody-dependent cell-mediated cytotoxicity. According to the
invention, a single antibody may have mutations in any one or more
of the CDRs or framework regions of the variable domain or in the
constant region.
[0156] In a process known as "germlining", certain amino acids in
the VH and VL sequences can be mutated to match those found
naturally in germline VH and VL sequences. In particular, the amino
acid sequences of the framework regions in the VH and VL sequences
can be mutated to match the germline sequences to reduce the risk
of immunogenicity when the antibody is administered. As used
herein, the term "germline" refers to the nucleotide sequences and
amino acid sequences of the antibody genes and gene segments as
they are passed from parents to offspring via the germ cells. This
germline sequence is distinguished from the nucleotide sequences
encoding antibodies in mature B cells which have been altered by
recombination and hypermutation events during the course of B cell
maturation. An antibody that "utilizes" a particular germline has a
nucleotide or amino acid sequence that most closely aligns with
that germline nucleotide sequence or with the amino acid sequence
that it specifies. Such antibodies frequently are mutated compared
with the germline sequence. Germline DNA sequences for human VH and
VL genes are known in the art (see e.g., the "Vbase" human germline
sequence database; see also Kabat, E. A., et al., 1991, Sequences
of Proteins of Immunological Interest, Fifth Edition, U.S.
Department of Health and Human Services, NIH Publication No.
91-3242; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox
et al., Eur. J. Immunol. 24:827-836, 1994.
[0157] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant region of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution is to
eliminate asparagine-glycine pairs, which form potential
deamidation sites, by altering one or both of the residues. In
another example, the C-terminal lysine of the heavy chain of an
EFNA4 antibody of the invention can be cleaved. In various aspects
of the invention, the heavy and light chains of the EFNA4
antibodies may optionally include a signal sequence.
[0158] Once DNA fragments encoding the VH and VL segments of the
present invention are obtained, these DNA fragments can be further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes, or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively
linked to another DNA fragment encoding another protein, such as an
antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined such that the amino acid
sequences encoded by the two DNA fragments remain in-frame.
[0159] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG2 constant region. The IgG constant
region sequence can be any of the various alleles or allotypes
known to occur among different individuals, such as Gm(1), Gm(2),
Gm(3), and Gm(17). These allotypes represent naturally occurring
amino acid substitution in the IgG1 constant regions. For a Fab
fragment heavy chain gene, the VH-encoding DNA can be operatively
linked to another DNA molecule encoding only the heavy chain CH1
constant region. The CH1 heavy chain constant region may be derived
from any of the heavy chain genes.
[0160] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region. The kappa constant region may be any of the
various alleles known to occur among different individuals, such as
Inv(1), Inv(2), and Inv(3). The lambda constant region may be
derived from any of the three lambda genes.
[0161] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker such that the VH and VL sequences can be expressed as a
contiguous single-chain protein, with the VL and VH regions joined
by the flexible linker (See e.g., Bird et al., 1988, Science
242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., 1990, Nature 348:552-554. The
single chain antibody may be monovalent, if only a single VH and VL
are used, bivalent, if two VH and VL are used, or polyvalent, if
more than two VH and VL are used. Bispecific or polyvalent
antibodies may be generated that bind specifically to EFNA4 and to
another molecule.
[0162] In another aspect of the invention, a fusion antibody or
immunoadhesin may be made that includes all or a portion of an
EFNA4 antibody of the invention linked to another polypeptide. In
another aspect, only the variable domains of the EFNA4 antibody are
linked to the polypeptide. In another aspect, the VH domain of an
EFNA4 antibody is linked to a first polypeptide, while the VL
domain of an EFNA4 antibody is linked to a second polypeptide that
associates with the first polypeptide in a manner such that the VH
and VL domains can interact with one another to form an antigen
binding site. In another aspect, the VH domain is separated from
the VL domain by a linker such that the VH and VL domains can
interact with one another. The VH-linker-VL antibody is then linked
to the polypeptide of interest. In addition, fusion antibodies can
be created in which two (or more) single-chain antibodies are
linked to one another. This is useful if one wants to create a
divalent or polyvalent antibody on a single polypeptide chain, or
if one wants to create a bispecific antibody.
[0163] Other modified antibodies may be prepared using EFNA4
antibody encoding nucleic acid molecules. For instance, "Kappa
bodies" (Ill et al., Protein Eng. 10:949-57, 1997), "Minibodies"
(Martin et al., EMBO J., 13:5303-9, 1994), "Diabodies" (Holliger et
al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993), or "Janusins"
(Traunecker et al., EMBO J. 10:3655-3659, 1991 and Traunecker et
al., Int. J. Cancer (Suppl.) 7:51-52, 1992) may be prepared using
standard molecular biological techniques following the teachings of
the specification.
[0164] Bispecific antibodies or antigen binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
Clin. Exp. Immunol. 79:315-321, 1990, Kostelny et al., J. Immunol.
148:1547-1553, 1992. In addition, bispecific antibodies may be
formed as "diabodies" or "Janusins." In some aspects of the
invention, a bispecific antibody binds to two different epitopes of
EFNA4. In other aspects, modified antibodies described above are
prepared using one or more of the variable domains or CDR regions
from the EFNA4 antibodies provided herein.
[0165] For use in preparation of antibody-drug conjugates, EFNA4
antibodies described herein may be substantially pure, i.e., at
least 50% pure (i.e., free from contaminants), more preferably, at
least 90% pure, more preferably, at least 95% pure, yet more
preferably, at least 98% pure, and most preferably, at least 99%
pure.
[0166] Table 2 provides the amino acid (protein) sequences and
associated nucleic acid (DNA) sequences of humanized anti-EFNA4
antibodies of the present invention. The CDRs of huE5 VH, huE5 VL,
huE15 VH, and huE15 VL, as defined by Kabat, are underlined. The
CDRs of huE22 VH, huE22 VL, huE47 VH, and huE47 VL, as defined by
Kabat and by Chothia, are set forth as separate sequences.
TABLE-US-00002 TABLE 2 Sequences of humanized anti-EFNA4
antibodies. SEQ ID NO Description Sequences 5 huE5 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTVTTYGVDWVRQAPG Protein
KGLEWLGVIWGGGSTNYNSALKSRFTISRDNSKNTLYLQMNS
LRAEDTAVYYCASDWAYWGQGTLVTVSS 6 huE5 VH
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCT DNA
GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
GTCACTACTTATGGTGTGGACTGGGTCCGCCAAGCTCCAGGG
AAGGGGCTGGAGTGGTTAGGTGTAATATGGGGTGGTGGAAGC
ACAAATTATAATAGCGCTTTGAAGAGCCGATTCACCATCTCC
AGAGACAACTCCAAGAACACCCTGTATCTGCAAATGAACAGT
CTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGTGAT
TGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTC 7 huE5 VL
DIQMTQSPSSLSASVGDRVTITCRASQNVGTNVAWFQQKPGK Protein
APKSLIHSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYKRYPYTFGGGTKLEIK
8 huE5 VL GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCT DNA
GTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGAAT
GTGGGTACAAATGTAGCCTGGTTTCAGCAGAAACCAGGGAAA
GCCCCTAAGTCCCTGATCCATTCGGCATCCTACCGTTACAGT
GGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAT
TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCA
ACTTACTATTGTCAGCAATATAAGAGGTATCCGTACACGTTC
GGAGGGGGGACCAAGCTGGAAATAAAAC 9 huE15 VH
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQAPG Protein
KGLEWVATISSGGTYTYYPDSVKGRFKISRDNAKNSLYLQMN
SLRAEDTAVYYCTRHDPNDGYYFLFAYWGQGTLVTVSS 10 huE15 VH
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCT DNA
GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
TTCAGTACCTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGG
AAGGGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTACT
TACACATACTACCCAGACTCAGTGAAGGGCCGATTCAAAATC
TCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC
AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACAAGA
CATGACCCCAATGATGGTTACTACTTCCTGTTTGCTTACTGG
GGCCAGGGGACTCTGGTCACTGTCTCTTC 11 huE15 VL
EIVLTQSPGTLSLSPGERATLSCKASQSVGNNVAWYQQKPGQ Protein
APRLLIYYASNRYTGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQHYSSPLTFGAGTKLEIK
12 huE15 VL GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCT DNA
CCAGGGGAAAGAGCCACCCTCTCCTGCAAGGCCAGTCAGAGT
GTTGGCAACAATGTAGCTTGGTACCAGCAGAAACCTGGCCAG
GCTCCCAGGCTCCTCATCTACTATGCATCCAATAGGTATACA
GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGAC
TTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCA
GTGTATTACTGTCAACAGCATTATAGCTCTCCGCTCACGTTC
GGTGCTGGGACCAAGCTGGAGATCAAAC 13 huE22 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPG Protein
QGLEWMGWIYPGNFNTKYNERFKGRVTMTTDTSTSTAYMELR
SLRSDDTAVYYCAREDGSPYYAMDYWGQGTSVTVSS 14 huE22 VH
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCT DNA
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACC
TTTACCGGCTATTACATCCACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGATGGGATGGATCTACCCTGGCAATTTT
AACACAAAATATAACGAGCGGTTCAAGGGCAGAGTCACCATG
ACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGG
AGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GAGGATGGTAGCCCCTACTATGCTATGGACTACTGGGGTCAA GGAACCTCAGTCACCGTCTCCTCA
15 huE22 VH CDR1 GYYIH Protein-Kabat 16 huE22 VH CDR1 GYTFTGY
Protein-Chothia 17 huE22 VH CDR1 GGCTATTACATCCAC DNA-Kabat 18 huE22
VH CDR1 GGTTACACCTTTACCGGCTAT DNA-Chothia 19 huE22 VH CDR2
WIYPGNFNTKYNERFKG Protein-Kabat 20 huE22 VH CDR2 YPGNFN
Protein-Chothia 21 huE22 VH CDR2
TGGATCTACCCTGGCAATTTTAACACAAAATATAACGAGCGG DNA-Kabat TTCAAGGGC 22
huE22 VH CDR2 TACCCTGGCAATTTTAAC DNA-Chothia 23 huE22 VH CDR3
EDGSPYYAMDY Protein-Kabat and Chothia 24 huE22 VH CDR3
GAGGATGGTAGCCCCTACTATGCTATGGACTAC DNA-Kabat and Chothia 25 huE22 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPG Protein-HulgG1
QGLEWMGWIYPGNFNTKYNERFKGRVTMTTDTSTSTAYMELR
SLRSDDTAVYYCAREDGSPYYAMDYWGQGTSVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG 26 huE22 HC
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCT DNA
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACC
TTTACCGGCTATTACATCCACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGATGGGATGGATCTACCCTGGCAATTTT
AACACAAAATATAACGAGCGGTTCAAGGGCAGAGTCACCATG
ACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGG
AGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GAGGATGGTAGCCCCTACTATGCTATGGACTACTGGGGTCAA
GGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTCGAGCAAGAGCACCTCTGGG
GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAGCCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG
GGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC
ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG
GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG
TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC
CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG
AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGT 27 huE22 VL
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLYWYL Protein
QKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQATHVPWTFGGGTKVEIK 28 huE22 VL
GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACC DNA
CCTGGACAGCCGGCCTCCATCTCCTGCCGGTCTAGTCAGAGC
CTCGTGCATAGTAATGGAAACACCTTTTTGTATTGGTACCTG
CAGAAGCCAGGCCAGTCTCCACAGCTCCTAATCTATAGAGTT
TCCAACCGGTTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGC
GGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAG
GCTGAGGATGTTGGGGTTTATTACTGCTTTCAAGCTACACAT
GTTCCGTGGACGTTCGGTGGAGGCACCAAAGTGGAAATCAAA 29 huE22 VL CDR1
RSSQSLVHSNGNTFLY Protein-Kabat 30 huE22 VL CDR1 QSLVHSNGNTF
Protein-Chothia 31 huE22 VL CDR1
CGGTCTAGTCAGAGCCTCGTGCATAGTAATGGAAACACCTTT DNA-Kabat TTGTAT 32
huE22 VL CDR1 CAGAGCCTCGTGCATAGTAATGGAAACACCTTT DNA-Chothia 33
huE22 VL CDR2 RVSNRFS Protein-Kabat and Chothia 34 huE22 VL CDR2
AGAGTTTCCAACCGGTTCTCT DNA-Kabat and Chothia 35 huE22 VL CDR3
FQATHVPWT Protein-Kabat and Chothia 36 huE22 VL CDR3
CAAGCTACACATGTTCCGTGGACG DNA-Kabat and Chothia 37 huE22 LC
DIVMTQTPLSLSVTPGQPASISCRSSQSLVHSNGNTFLYWYL Protein-Kappa
QKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCFQATHVPWTFGGGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 38 huE22 LC
GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACC DNA
CCTGGACAGCCGGCCTCCATCTCCTGCCGGTCTAGTCAGAGC
CTCGTGCATAGTAATGGAAACACCTTTTTGTATTGGTACCTG
CAGAAGCCAGGCCAGTCTCCACAGCTCCTAATCTATAGAGTT
TCCAACCGGTTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGC
GGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAG
GCTGAGGATGTTGGGGTTTATTACTGCTTTCAAGCTACACAT
GTTCCGTGGACGTTCGGTGGAGGCACCAAAGTGGAAATCAAA
CGGACTGTGGCTGCACCAAGTGTCTTCATCTTCCCGCCATCT
GATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC
ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC
ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC
ACAAAGAGCTTCAACAGGGGAGAGTGT 39 huE47 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTYFYMNWVRQAPG Protein
QGLEWVGQINPNNGGTAYAQKFQGRVTMTRDTSTSTVYMELS
SLRSEDTAVYYCARWVGTHYFDYWGQGTTLTVSS 40 huE47 VH
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT DNA
GGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACC
TTCACTTACTTCTATATGAACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGGTGGGACAAATCAACCCTAATAATGGT
GGCACAGCCTACGCACAGAAGTTCCAGGGCAGAGTCACCATG
ACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGC
AGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA
TGGGTCGGGACTCACTACTTTGACTACTGGGGCCAAGGCACC ACTCTCACAGTCTCCTCC 41
huE47 VH CDR1 YFYMN Protein-Kabat 42 huE47 VH CDR1 GYTFTYF
Protein-Chothia 43 huE47 VH CDR1 TACTTCTATATGAAC DNA-Kabat 44 huE47
VH CDR1 GGATACACCTTCACTTACTTC DNA-Chothia 45 huE47 VH CDR2
QINPNNGGTAYAQKFQG Protein-Kabat 46 huE47 VH CDR2 NPNNGGT
Protein-Chothia 47 huE47 VH CDR2
CAAATCAACCCTAATAATGGTGGCACAGCCTACGCACAGAAG
DNA-Kabat TTCCAGGGC 48 huE47 VH CDR2 AACCCTAATAATGGTGGCACA
DNA-Chothia 49 huE47 VH CDR3 WVGTHYFDY Protein-Kabat and Chothia 50
huE47 VH CDR3 TGGGTCGGGACTCACTACTTTGACTAC DNA-Kabat and Chothia 51
huE47 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTYFYMNWVRQAPG Protein-Human
QGLEWVGQINPNNGGTAYAQKFQGRVTMTRDTSTSTVYMELS IGg1
SLRSEDTAVYYCARWVGTHYFDYWGQGTTLTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 52 huE47 HC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT DNA
GGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACC
TTCACTTACTTCTATATGAACTGGGTGCGACAGGCCCCTGGA
CAAGGGCTTGAGTGGGTGGGACAAATCAACCCTAATAATGGT
GGCACAGCCTACGCACAGAAGTTCCAGGGCAGAGTCACCATG
ACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGC
AGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA
TGGGTCGGGACTCACTACTTTGACTACTGGGGCCAAGGCACC
ACTCTCACAGTCTCCTCCGCCTCCACCAAGGGCCCATCGGTC
TTCCCCCTGGCGCCCTCGAGCAAGAGCACCTCTGGGGGCACA
GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG
CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC
CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC
CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA
CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT 53 huE47 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSSSYTYIHWYQQ Protein
KPGQAPRLLINFASNLESGIPARFSGSGSGTDFTLTISSLEP
EDFAVYYCQHSWEIPPTFGGGTKLEIK 54 huE47 VL
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCT DNA
CCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGT
GTTAGCAGCTCTAGCTATACTTACATTCACTGGTACCAACAG
AAACCTGGCCAGGCTCCCAGGCTCCTCATCAATTTTGCATCC
AACTTGGAAAGTGGCATCCCAGCCAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCT
GAAGATTTTGCAGTTTATTACTGTCAGCACAGTTGGGAGATT
CCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 55 huE47 VL CDR1
RASQSVSSSSYTYIH Protein-Kabat 56 huE47 VL CDR1 SSSYTYIH
Protein-Chothia 57 huE47 VL CDR1
AGGGCCAGTCAGAGTGTTAGCAGCTCTAGCTATACTTACATT DNA-Kabat CAC 58 huE47
VL CDR1 AGCTCTAGCTATACTTACATTCAC DNA-Chothia 59 huE47 VL CDR2
FASNLES Protein-Kabat and Chothia 60 huE47 VL CDR2
TTTGCATCCAACTTGGAAAGT DNA-Kabat and Chothia 61 huE47 VL CDR3
QHSWEIPPT Protein-Kabat and Chothia 62 huE47 VL CDR3
CAGCACAGTTGGGAGATTCCTCCGACG DNA-Kabat and Chothia 63 huE47 LC
EIVLTQSPATLSLSPGERATLSCRASQSVSSSSYTYIHWYQQ Protein-Human
KPGQAPRLLINFASNLESGIPARFSGSGSGTDFTLTISSLEP Kappa
EDFAVYYCQHSWEIPPTFGGGTKLEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 64 huE47 LC
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCT DNA
CCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGT
GTTAGCAGCTCTAGCTATACTTACATTCACTGGTACCAACAG
AAACCTGGCCAGGCTCCCAGGCTCCTCATCAATTTTGCATCC
AACTTGGAAAGTGGCATCCCAGCCAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCT
GAAGATTTTGCAGTTTATTACTGTCAGCACAGTTGGGAGATT
CCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG
ACTGTGGCTGCACCAAGTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG
GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGT
[0167] II.B. Linkers
[0168] Anti-EFNA4 antibody-drug conjugates of the present invention
can be prepared using a linker to link or conjugate a drug to an
anti-EFNA4 antibody. In particular aspects of the invention, the
linker of EFNA4 antibody-drug conjugates of the invention includes,
but is not limited to, 4-(4'acetylphenoxy)butanoic acid
(AcBut).
[0169] The linker molecule may be stable (non-cleavable) or
hydrolysable (cleavable), whereby it is released following cellular
entry. The major mechanisms by which the drug is cleaved from the
antibody include hydrolysis in the acidic pH of the lysosomes
(hydrazones, acetals, and cis-aconitate-like amides), peptide
cleavage by lysosomal enzymes (the cathepsins and other lysosomal
enzymes), and reduction of disulfides. As a result of these varying
mechanisms for cleavage, mechanisms of linking the drug to the
antibody also vary widely and any suitable linker can be used.
[0170] An example of a suitable conjugation procedure relies on the
conjugation of hydrazides and other nucleophiles to the aldehydes
generated by oxidation of the carbohydrates that naturally occur on
antibodies. Hydrazone-containing conjugates can be made with
introduced carbonyl groups that provide the desired drug-release
properties. Conjugates can also be made with a linker that has a
disulfide at one end, an alkyl chain in the middle, and a hydrazine
derivative at the other end. The anthracyclines are one example of
cytotoxins that can be conjugated to antibodies using this
technology.
[0171] Linkers containing functional groups other than hydrazones
have the potential to be cleaved in the acidic milieu of the
lysosomes. For example, conjugates can be made from thiol-reactive
linkers that contain a site other than a hydrazone that is
cleavable intracellularly, such as esters, amides, and
acetals/ketals. Camptothecin is one cytotoxic agent that can be
conjugated using these linkers. Ketals made from a 5 to 7-member
ring ketone and that has one of the oxygens attached to the
cytotoxic agent and the other to a linker for antibody attachment
also can be used. The anthracyclines are also an example of a
suitable cytotoxin for use with these linkers.
[0172] Another example of a class of pH sensitive linkers are the
cis-aconitates, which have a carboxylic acid juxtaposed to an amide
bond. The carboxylic acid accelerates amide hydrolysis in the
acidic lysosomes. Linkers that achieve a similar type of hydrolysis
rate acceleration with several other types of structures can also
be used. The maytansinoids are an example of a cytotoxin that can
be conjugated with linkers attached at C-9.
[0173] Another potential release method for drug conjugates is the
enzymatic hydrolysis of peptides by the lysosomal enzymes. In one
example, a peptide is attached via an amide bond to
para-aminobenzyl alcohol and then a carbamate or carbonate is made
between the benzyl alcohol and the cytotoxic agent. Cleavage of the
peptide leads to the collapse, or self-immolation, of the
aminobenzyl carbamate or carbonate. The cytotoxic agents
exemplified with this strategy include anthracyclines, taxanes,
mitomycin C, and the auristatins. In one example, a phenol can also
be released by collapse of the linker instead of the carbamate. In
another variation, disulfide reduction is used to initiate the
collapse of a para-mercaptobenzyl carbamate or carbonate.
[0174] Many of the cytotoxic agents conjugated to antibodies have
little, if any, solubility in water and that can limit drug loading
on the conjugate due to aggregation of the conjugate. One approach
to overcoming this is to add solublizing groups to the linker.
Conjugates made with a linker consisting of PEG and a dipeptide can
been used, including those having a PEG di-acid, thiol-acid, or
maleimide-acid attached to the antibody, a dipeptide spacer, and an
amide bond to the amine of an anthracycline or a duocarmycin
analogue. Another example is a conjugate prepared with a
PEG-containing linker disulfide bonded to a cytotoxic agent and
amide bonded to an antibody. Approaches that incorporate PEG groups
may be beneficial in overcoming aggregation and limits in drug
loading.
[0175] In some aspects of the invention, the linkers for the
preparation of the antibody-drug conjugates of the present
invention include linkers having the formula:
(CO-Alk.sup.1-Sp.sup.1-Ar-Sp.sup.2-Alk.sup.2-C(Z.sup.1)=Q-Sp)
wherein [0176] Alk.sup.1 and Alk.sup.2 are independently a bond or
branched or unbranched (C.sub.1-C.sub.10) alkylene chain; [0177]
Sp.sup.1 is a bond, --S--, --O--, --CONH--, --NHCO--, --NR'--,
--N(CH.sub.2CH.sub.2).sub.2N--, or --X--Ar'--Y--(CH.sub.2).sub.n--Z
wherein X, Y, and Z are independently a bond, --NR'--, --S--, or
--O--, with the proviso that when n=0, then at least one of Y and Z
must be a bond and Ar' is 1,2-, 1,3-, or 1,4-phenylene optionally
substituted with one, two, or three groups of (C.sub.1-C.sub.5)
alkyl, (C.sub.1-C.sub.4) alkoxy, (C.sub.1-C.sub.4) thioalkoxy,
halogen, nitro, --COOR', --CONHR', --(CH.sub.2).sub.nCOOR',
--S(CH.sub.2).sub.nCOOR', --O(CH.sub.2).sub.nCONHR', or
--S(CH.sub.2).sub.nCONHR', with the proviso that when Alk' is a
bond, Sp.sup.1 is a bond; [0178] n is an integer from 0 to 5;
[0179] R' is a branched or unbranched (C.sub.1-C.sub.5) chain
optionally substituted by one or two groups of --OH,
(C.sub.1-C.sub.4) alkoxy, (C.sub.1-C.sub.4) thioalkoxy, halogen,
nitro, (C.sub.1-C.sub.3) dialkylamino, or (C.sub.1-C.sub.3)
trialkylammonium --A.sup.- where A.sup.- is a pharmaceutically
acceptable anion completing a salt; [0180] Ar is 1,2-, 1,3-, or
1,4-phenylene optionally substituted with one, two, or three groups
of (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.5) alkoxy,
(C.sub.1-C.sub.4) thioalkoxy, halogen, nitro, --COOR', --CONHR',
--O(CH.sub.2).sub.nCOOR', --S(CH.sub.2).sub.nCOOR',
--O(CH.sub.2).sub.nCONHR', or --S(CH.sub.2).sub.nCONHR' wherein n
and R' are as hereinbefore defined or a 1,2-, 1,3-, 1,4-, 1,5-,
1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene or
[0180] ##STR00001## [0181] with each naphthylidene or phenothiazine
optionally substituted with one, two, three, or four groups of
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.5) alkoxy,
(C.sub.1-C.sub.4) thioalkoxy, halogen, nitro, --COOR', --CONHR',
--O(CH.sub.2).sub.nCOOR', --S(CH.sub.2).sub.nCOOR', or
--S(CH.sub.2).sub.nCONHR' wherein n and R' are as defined above,
with the proviso that when Ar is phenothiazine, Sp.sup.1 is a bond
only connected to nitrogen; [0182] Sp.sup.2 is a bond, --S--, or
--O--, with the proviso that when Alk.sup.2 is a bond, Sp.sup.2 is
a bond, [0183] Z.sup.1 is H, (C.sub.1-C.sub.5) alkyl, or phenyl
optionally substituted with one, two, or three groups of
(C.sub.1-C.sub.5) alkyl, (C.sub.1-C.sub.5) alkoxy,
(C.sub.1-C.sub.4) thioalkoxy, halogen, nitro, --COOR', --ONHR',
--O(CH.sub.2).sub.nCOOR', --S(CH.sub.2).sub.nCOOR',
--O(CH.sub.2).sub.nCONHR', or --S(CH.sub.2).sub.nCONHR' wherein n
and R' are as defined above; [0184] Sp is a straight or
branched-chain divalent or trivalent (C.sub.1-C.sub.18) radical,
divalent or trivalent aryl or heteroaryl radical, divalent or
trivalent (C.sub.3-C.sub.18) cycloalkyl or heterocycloalkyl
radical, divalent or trivalent aryl- or heteroaryl-aryl
(C.sub.1-C.sub.18) radical, divalent or trivalent cycloalkyl- or
heterocycloalkyl-alkyl (C.sub.1-C.sub.18) radical or divalent or
trivalent (C.sub.2-C.sub.18) unsaturated alkyl radical, wherein
heteroaryl is preferably furyl, thienyl, N-methylpyrrolyl,
pyridinyl, N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl,
isoquinolyl, N-methylcarbazoyl, aminocourmarinyl, or phenazinyl and
wherein if Sp is a trivalent radical, Sp may be additionally
substituted by lower (C.sub.1-C.sub.5) dialkylamino, lower
(C.sub.1-C.sub.5) alkoxy, hydroxy, or lower (C.sub.1-C.sub.5)
alkylthio groups; and [0185] Q is .dbd.NHNCO--, .dbd.NHNCS--,
.dbd.NHNCONH--, .dbd.NHNCSNH--, or .dbd.NHO--.
[0186] Preferably, Alk.sup.1 is a branched or unbranched
(C.sub.1-C.sub.10) alkylene chain; Sp' is a bond, --S--, --O--,
--CONH--, --NHCO--, or --NR' wherein R' is as hereinbefore defined,
with the proviso that when Alk' is a bond, Sp.sup.1 is a bond;
[0187] Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted
with one, two, or three groups of (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.5) alkoxy, (C.sub.1-C.sub.4) thioalkoxy, halogen,
nitro, --COOR', --CONHR', --O(CH.sub.2).sub.nCOOR',
--S(CH.sub.2).sub.nCOOR', --O(CH.sub.2).sub.nCONHR', or
--S(CH.sub.2).sub.nCONHR' wherein n and R' are as hereinbefore
defined, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-,
2,6-, or 2,7-naphthylidene each optionally substituted with one,
two, three, or four groups of (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.5) alkoxy, (C.sub.1-C.sub.4) thioalkoxy, halogen,
nitro, --COOR', --CONHR', --O(CH.sub.2).sub.nCOOR',
--S(CH.sub.2).sub.nCOOR', --O(CH.sub.2).sub.nCONHR', or
--S(CH.sub.2).sub.nCONHR'. [0188] Z.sup.1 is (C.sub.1-C.sub.5)
alkyl, or phenyl optionally substituted with one, two, or three
groups of (C.sub.1-C.sub.5) alkyl, (C.sub.1-C.sub.4) alkoxy,
(C.sub.1-C.sub.4) thioalkoxy, halogen, nitro, --COOR', --CONHR',
--O(CH.sub.2).sub.nCOOR', --S(CH.sub.2).sub.nCOOR',
--O(CH.sub.2).sub.nCONHR', or --S(CH.sub.2).sub.nCONHR'; Alk.sup.2
and Sp.sup.2 are together a bond; and Sp and Q are as immediately
defined above.
[0189] U.S. Pat. No. 5,773,001, which is incorporated herein by
reference in its entirety, discloses linkers that may be used with
nucleophilic drugs, particularly hydrazides and related
nucleophiles, prepared from the calicheamicins. These linkers are
especially useful in those cases where better activity is obtained
when the linkage formed between the drug and the linker is
hydrolysable. These linkers contain two functional groups,
including (1) a group for reaction with an antibody (e.g.,
carboxylic acid), and (2) a carbonyl group (e.g., an aldehyde or a
ketone) for reaction with a drug. The carbonyl groups may react
with a hydrazide group on the drug to form a hydrazone linkage.
This linkage is cleavable hydrolysable, allowing for release of the
therapeutic agent from the conjugate after binding to the target
cells. In some aspects of the invention, the hydrolysable linker
used is 4-(4-acetylphenoxy) butanoic acid (AcBut). In other aspects
of the invention, antibody-drug conjugates can be prepared using
(3-Acetylphenyl) acetic acid (AcPAc) or
4-mercapto-4-methyl-pentanoic acid (Amide) as the linker
molecule.
[0190] N-hydroxysuccinimide (OSu) esters or other comparably
activated esters can be used to generate the activated hydrolyzable
linker-drug moiety. Examples of other suitable activating esters
include NHS (N-hydroxysuccinimide), sulfo-NHS (sulfonated NHS), PFP
(pentafluorophenyl), TFP (tetrafluorophenyl), and DNP
(dinitrophenyl).
[0191] In some aspects of the invention, the antibody-drug
conjugates are prepared by reacting calicheamicin or derivatives
thereof, the AcBut linker and an anti-EFNA4 antibody of the present
invention. See e.g., U.S. Pat. No. 5,773,001. The AcBut linker
produces conjugates that are substantially stable in circulation,
releasing an estimated 2% of the calicheamicin per day when assayed
at 37.degree. C. in human plasma in vitro. The conjugates release
the calicheamicin in the acidic lysosomes.
[0192] In some aspects of the invention, the AcBut-CM moiety can be
generated using methods and processes described in the art, such as
PCT International Publication No. WO 08/147765 and in U.S. Pat. No.
8,273,862, which are incorporated herein by reference in their
entirety.
[0193] In some aspects of the invention, the AcBut-CM moiety can be
generated using an improved synthesis process, as described in U.S.
Provisional Application No. 61/899,682, which is incorporated
herein by reference in its entirety. The method for synthesizing a
linker intermediate (compound 10) is described as follows:
##STR00002##
[0194] In Scheme 1, compound 1 and compound 2 are reacted in
2-methyltetrahydrofuran (2-MeTHF) and tetrabutylammonium fluoride
(Bu.sub.4NF). A solution of calcium chloride dihydrate in water is
added, and the lower aqueous phase removed after stirring. To the
upper organic phase is added methanol and 3 equiv. of NaOH in
water. The reaction mixture is stirred until complete consumption
of the intermediate ester 3 is observed. The reaction is cooled to
15.degree. C. and 2-methyltetrahydrofuran is added followed by
water. Concentrated HCl is added slowly, maintaining the reaction
in the range 15-30.degree. C. Acid 4 is yielded from the organic
layer.
##STR00003##
[0195] In Scheme 2, compound 4 is charged with a suitable organic
solvent such as tetrahydrofuran (THF) and an azole activating
agent, such as carbonyl diimidazole (CDI), forming intermediate 5.
Other azole activating agents may be used, for example thiocarbonyl
diimidazole; carbonyl bis-pyrazole wherein each pyrazole optionally
substituted with from one to three (C.sub.1-C.sub.6) alkyl groups;
carbonyl bis-1,2,3-triazole; carbonyl bis-benzotriazole, and
carbonyl bis-1,2,4-triazole, which would thereby form intermediate
compounds analogous to compound 5 but comprising a different azole
moiety other than imidazolyl. Intermediate 5 is reacted with
hydrazine, preferably an aqueous source of hydrazine such as
hydrazine monohydrate, yielding intermediate 6. Intermediate 6 is
described in PCT International Publication No. WO 08/147765, which
is incorporated herein by reference in its entirety.
##STR00004##
[0196] In Scheme 3, intermediate 6 is reacted with intermediate 7
in an inert (non-reactive) solvent (such as methanol (MeOH)),
optionally with an acidic catalyst (such as acetic acid (HOAc)) to
yield intermediate 8.
##STR00005##
[0197] In Scheme 4, intermediate 8 is deprotected by using a strong
acid optionally under heat, such as trifluoroacetic acid (TFA)
under heat, to form intermediate 9. Other strong acids may be used
instead of trifluoroacetic acid, for example sulfuric acid.
##STR00006##
[0198] In Scheme 5, intermediate 9 is converted to linker
intermediate 10. Intermediate 9 can be converted to a linker
intermediate 10 as is described in the art, such as in U.S. Pat.
No. 8,273,862, which is incorporated herein by reference in its
entirety. Preferably, however, as depicted in Scheme 5,
intermediate 9 is reacted with a tertiary amine base such as
triethylamine (TEA) and with trimethylacetyl chloride (PivCl) in
the presence of an inert solvent such as tetrahydrofuran.
Subsequently, N-hydroxysuccinimide (OSu) is introduced to provide
the linker intermediate 10.
[0199] The process for synthesizing linker intermediate 10
described herein above provides improvements over those processes
described in PCT International Publication No. WO 08/147765 and in
U.S. Pat. No. 8,273,862, because the process described herein above
avoids using methylene chloride and the safety measures taken
therewith, and offers better yields of linker intermediate 10.
[0200] Subsequent to synthesis of linker intermediate 10, linker
intermediate 10 may be conjugated to a calicheamicin molecule as is
described in the prior art, for example in U.S. Pat. No. 8,273,862.
Preferably, however the linker intermediate 10 is combined with a
calicheamicin in the presence of a carbodiimide, which improves the
yield of the resulting calicheamicin derivative. Examples of
carbodiimides that can be used include, but are not limited to,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC);
N,N'-dicyclohexyl carbodiimide (DCC); N,N'-diisopropyl carbodiimide
(DIC); N-cylcohexyl-N'-(2-morpholinoethyl) carbodiimide;
N-cylcohexyl-N'-[2-(4-methylmorpholin-4-ium-4-yl)ethyl]
carbodiimide tosylate;
N-cylcohexyl-N'-[4-(diethylmethylammonio)cyclohexyl] carbodiimide
tosylate;
N,N'-bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]carbodiimide; and
N-benzyl-N'-isopropylcarbodiimide.
[0201] The calicheamicin derivative resulting from the conjugation
of linker intermediate 10 to calicheamicin can be purified and
isolated prior to reaction with a monoclonal antibody (e.g.
anti-EFNA4 antibody). The purification and isolation may take place
as is described previously in U.S. Pat. No. 8,273,862. Or the
purification may take place by using a reversed phase high
performance liquid chromatography purification protocol. The
reversed phase high performance liquid chromatography purification
protocol for purification of 10 may comprise elution with phases
comprising aqueous and organic mixtures, which phases range in pH
from about 4 to about 6. For example, a mobile phase consisting of
55% 20 mM sodium acetate, pH 5 and 45% acetonitrile is an
aqueous/organic mobile phase that can be used for the reversed
phase high performance liquid chromatography purification. The
purification with reversed phase high performance liquid
chromatography purification protocol may be followed by a solid
phase extraction protocol.
[0202] In other aspects of the invention, the linker can be a
dipeptide linker, such as a valine-citrulline (val-cit), a
phenylalanine-lysine (phe-lys) linker, or
maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc)
linker. In another aspect, the linker is
Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(smcc). Sulfo-smcc conjugation occurs via a maleimide group which
reacts with sulfhydryls (thiols, --SH), while its Sulfo-NHS ester
is reactive toward primary amines (as found in Lysine and the
protein or peptide N-terminus). Further, the linker may be
maleimidocaproyl (mc).
[0203] Representative linkers useful for conjugation of
radioisotopes include diethylenetriamine pentaacetate
(DTPA)-isothiocyanate, succinimidyl 6-hydrazinium nicotinate
hydrochloride (SHNH), and hexamethylpropylene amine oxime (HMPAO)
(Bakker et al. (1990) J. Nucl. Med. 31: 1501-1509, Chattopadhyay et
al. (2001) Nucl. Med. Biol. 28: 741-744, Dewanjee et al. (1994) J.
Nucl. Med. 35: 1054-63, Krenning et al. (1989) Lancet 1: 242-244,
Sagiuchi et al. (2001) Ann. Nucl. Med. 15: 267-270); U.S. Pat. No.
6,024,938). Alternatively, a targeting molecule may be derivatized
so that a radioisotope may be bound directly to it (Yoo et al.
(1997) J. Nucl. Med. 38: 294-300). Iodination methods are also
known in the art, and representative protocols may be found, for
example, in Krenning et al. (1989) Lancet 1:242-244 and in Bakker
et al. (1990) J. Nucl. Med. 31:1501-1509.
[0204] II.C. Drugs
[0205] Drugs useful in preparation of the disclosed EFNA4
antibody-drug conjugates include any substance having biological or
detectable activity, for example, therapeutic agents, detectable
labels, binding agents, etc., and prodrugs, which are metabolized
to an active agent in vivo. A drug may also be a drug derivative,
wherein a drug has been functionalized to enable conjugation with
an antibody of the invention. In accordance with the disclosed
methods, the drugs are used to prepare an antibody-drug conjugates
of the formula Ab-(L-D), wherein (a) Ab is an antibody, or
antigen-binding fragment thereof, that binds to EFNA4, and (b) L-D
is a linker-drug moiety, wherein L is a linker, and D is a drug.
The drug-to-antibody ratio (DAR) or drug loading, indicating the
number of drug (D) molecules conjugated per antibody, may be from
DAR 1 to 12. Thus, in aspects of the invention, an EFNA4
antibody-drug conjugate may have a DAR of 1, a DAR of 2, a DAR of
3, a DAR of 4, a DAR of 5, a DAR of 6, a DAR of 7, a DAR of 8, a
DAR of 9, a DAR of 10, a DAR of 11 or a DAR of 12. Thus, in aspects
of the invention, an EFNA4 antibody-drug conjugate may include one
drug molecule, or 2 drug molecules, or 3 drug molecules, or 4 drug
molecules, or 5 drug molecules, or 6 drug molecules, or 7 drug
molecules, or 8 drug molecules, or 9 drug molecules, or 10 drug
molecules, or 11 drug molecules, or 12 drug molecules. DAR can be
determined by various conventional means such as UV spectroscopy,
mass spectroscopy, ELISA assay, radiometric methods, hydrophobic
interaction chromatography (HIC), electrophoresis and HPLC.
[0206] Compositions, batches and/or formulations of antibody-drug
conjugate (ADC), of the formula Ab-(L-D), may include a plurality
of antibodies conjugated with a varying number of drug molecules,
from DAR 1 to 12.
[0207] In particular aspects of the invention, a composition,
batch, and/or formulation of antibody-drug conjugates may be
characterized by an average DAR in the range of about 1 to about
12, for example, an average DAR in the range of about 2 to about 4,
or an average DAR in the range of about 3 to about 5, or an average
DAR in the range of about 4 to about 6, or an average DAR in the
range of about 5 to about 7, or an average DAR in the range of
about 6 to about 8, or an average DAR in the range of about 7 to
about 9, or an average DAR in the range of about 8 to about 10, or
an average DAR in the range of about 9 to about 11. In some aspects
the compositions, batches and/or formulations of antibody-drug
conjugate may have an average DAR of about 1, or an average DAR of
about 2, an average DAR of about 3, or an average DAR of about 4,
or an average DAR of about 5, or an average DAR of about 6, or an
average DAR of about 7, or an average DAR of about 8, or an average
DAR of about 9, or an average DAR of about 10, or an average DAR of
about 11. As used in the foregoing ranges of average DAR, the term
"about" means +/-0.5%.
[0208] Moreover, a composition, batch, and/or formulation of
antibody-drug conjugates may be characterized by a preferred range
of average DAR, e.g., an average DAR in the range of about 3 to
about 5, an average DAR in the range of about 3 to about 4, or an
average DAR in the range of about 4 to about 5. Further, a
composition, batch, and/or formulation of antibody-drug conjugates
may be characterized by a preferred range of average DAR, e.g., an
average DAR in the range of 3 to 5, an average DAR in the range of
3 to 4, or an average DAR in the range of 4 to 5.
[0209] In some aspects of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by an average DAR of about 3.0, or an average DAR of 3.0, or an
average DAR of 3.1, or an average DAR of 3.2, or an average DAR of
3.3, or an average DAR of 3.4, or an average DAR of 3.5, or an
average DAR of 3.6, or an average DAR of 3.7, or an average DAR of
3.8, or an average DAR of 3.9. In another aspect of the invention,
a composition, batch, and/or formulation of antibody-drug
conjugates may be characterized by an average DAR of about 4.0, or
an average DAR of 4.0, or an average DAR of 4.1, or an average DAR
of 4.2, or an average DAR of 4.3, or an average DAR of 4.4, or an
average DAR of 4.5, or an average DAR of 4.6, or an average DAR of
4.7, or an average DAR of 4.8, or an average DAR of 4.9, or an
average DAR of 5.0.
[0210] In another aspect of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by an average DAR of 12 or less, an average DAR of 11 or less, an
average DAR of 10 or less, an average DAR of 9 or less, an average
DAR of 8 or less, an average DAR of 7 or less, an average DAR of 6
or less, an average DAR of 5 or less, an average DAR of 4 or less,
an average DAR of 3 or less, an average DAR of 2 or less or an
average DAR of 1 or less.
[0211] In other aspects of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by an average DAR of 11.5 or less, an average DAR of 10.5 or less,
an average DAR of 9.5 or less, an average DAR of 8.5 or less, an
average DAR of 7.5 or less, an average DAR of 6.5 or less, an
average DAR of 5.5 or less, an average DAR of 4.5 or less, an
average DAR of 3.5 or less, an average DAR of 2.5 or less, an
average DAR of 1.5 or less.
[0212] Compositions, batches and/or formulations of ADCs of the
formula Ab-(L-D), may be characterized by a DAR distribution. The
DAR distribution provides the percent or fraction of various ADC
species, e.g. DAR 1 to 12, that may be present in a composition,
batch, and/or formulation of ADCs. The DAR distribution of a
composition, batch, and/or formulation of ADCs may be determined by
methods known in the art, such as capillary iso-electric focusing
(cIEF).
[0213] In one aspect of the invention, the DAR distribution of a
composition, batch, and/or formulation of ADCs, of the formula
Ab-(L-D), may be characterized as a highly heterogeneous mixture of
ADCs with a broad DAR distribution, generally containing a broad
range of ADC species with DAR 1 to 12.
[0214] In another aspect of the invention, the DAR distribution of
a composition, batch, and/or formulation of ADCs may be
characterized as a highly homogeneous mixture with a narrow DAR
distribution, generally containing a narrow range of ADC species
having a particular DAR, such as DAR 3 to 5.
[0215] In particular aspects of the invention, a composition,
batch, and/or formulation of antibody-drug conjugates may be
characterized by having at least 50% antibody-drug conjugates
having a DAR from 3 to 5, or at least 55% antibody-drug conjugates
having a DAR from 3 to 5, or at least 60% antibody-drug conjugates
having a DAR from 3 to 5, or at least 65% antibody-drug conjugates
having a DAR from 3 to 5, or at least 70% antibody-drug conjugates
having a DAR from 3 to 5, or at least 75% antibody-drug conjugates
having a DAR from 3 to 5, or at least 80% antibody-drug conjugates
having a DAR from 3 to 5, or at least 85% antibody-drug conjugates
having a DAR from 3 to 5, or at least 90% antibody-drug conjugates
having a DAR from 3 to 5, or at least 95% antibody-drug conjugates
having a DAR from 3 to 5.
[0216] In another aspects of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by having 50% to 60% antibody-drug conjugates having a DAR from 3
to 5, or 60% to 70% antibody-drug conjugates having a DAR from 3 to
5, or 70% to 80% antibody-drug conjugates having a DAR from 3 to 5,
or 80% to 90% antibody-drug conjugates having a DAR from 3 to 5, or
90% to 100% antibody-drug conjugates having a DAR from 3 to 5. In
another aspects of the invention, a composition, batch, and/or
formulation of antibody-drug conjugates may be characterized by
having about 50%, or about 55%, or about 60%, or about 65%, or
about 70%, or about 75%, or about 80%, or about 85%, or about 90%,
or about 95%, or about 100% antibody-drug conjugates having a DAR
from 3 to 5.
[0217] For example, a therapeutic agent is an agent that exerts a
cytotoxic, cytostatic, and/or immunomodulatory effect on cancer
cells or activated immune cells. Examples of therapeutic agents
include cytotoxic agents, chemotherapeutic agents, cytostatic
agents, and immunomodulatory agents. Chemotherapeutic agents are
chemical compounds useful in the treatment of cancer.
[0218] Therapeutic agents are compositions that may be used to
treat or prevent a disorder in a subject in need thereof.
Therapeutic agents useful in the invention include anti-cancer
agents, i.e., agents having anti-cancer activity in
EFNA4-expressing cells such as cancer cells from breast cancer,
such as triple-negative breast cancer (TNBC); ovarian cancer;
colorectal cancer; leukemias, such as chronic lymphocytic leukemia
(CLL); liver cancer, such as hepatocellular carcinoma (HCC); and
lung cancer, such as non-small cell lung cancer (NSCLC) and small
cell lung cancer (SCLC).
[0219] Representative therapeutic agents include cytotoxins,
cytotoxic agents, and cytostatic agents. A cytotoxic effect refers
to the depletion, elimination and/or the killing of a target
cell(s). A cytotoxic agent refers to an agent that has a cytotoxic
and/or cytostatic effect on a cell. A cytostatic effect refers to
the inhibition of cell proliferation. A cytostatic agent refers to
an agent that has a cytostatic effect on a cell, thereby inhibiting
the growth and/or expansion of a specific subset of cells.
[0220] Additional representative therapeutic agents include
radioisotopes, chemotherapeutic agents, immunomodulatory agents,
anti-angiogenic agents, anti-proliferative agents, pro-apoptotic
agents, and cytolytic enzymes (e.g., RNAses). An agent may also
include a therapeutic nucleic acid, such as a gene encoding an
immunomodulatory agent, an anti-angiogenic agent, an
anti-proliferative agent, or a pro-apoptotic agent. These drug
descriptors are not mutually exclusive, and thus a therapeutic
agent may be described using one or more of the above-noted terms.
For example, selected radioisotopes are also cytotoxins.
Therapeutic agents may be prepared as pharmaceutically acceptable
salts, acids or derivatives of any of the above. Generally,
conjugates having a radioisotope as the drug are referred to as
radioimmunoconjugates and those having a chemotherapeutic agent as
the drug are referred to as chemoimmunoconjugates.
[0221] Examples of a cytotoxic agents include, but are not limited
to an anthracycline, an auristatin, CC-1065, a dolastatin, a
duocarmycin, an enediyne, a geldanamycin, a maytansine, a
puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin,
hemiasterlin, and stereoisomers, isosteres, analogs or derivatives
thereof. Plant toxins, other bioactive proteins, enzymes (i.e.,
ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic
therapy) can also be used.
[0222] The anthracyclines are derived from bacteria Strepomyces and
have been used to treat a wide range of cancers, such as leukemias,
lymphomas, breast, uterine, ovarian, and lung cancers. Exemplary
anthracyclines include, but are not limited to, daunorubicin,
doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin,
and mitoxantrone.
[0223] Dolastatins and their peptidic analogs and derivatives,
auristatins, are highly potent antimitotic agents that have been
shown to have anticancer and antifungal activity. See, e.g., U.S.
Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother.
42:2961-2965, (1998). Exemplary dolastatins and auristatins
include, but are not limited to, dolastatin 10, auristatin E,
auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl
Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl
Auristatin F or
N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE
(Monomethyl Auristatin E or
N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine),
5-benzoylvaleric acid-AE ester (AEVB). and other novel
[0224] In some aspects of the invention, auristatins described in
PCT International Publication No. WO 2013/072813, which is
incorporated herein by reference in its entirety, and methods of
producing those auristatins are used herein.
[0225] For example, the auristatin 0101,
(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrol-
idin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),
having the following structure:
##STR00007##
Additionally, the auristatin 8261,
2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phen-
ylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, having the
following structure:
##STR00008##
[0226] Duocarmycin and CC-1065 are DNA alkylating agents with
cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649, 1995.
Exemplary dolastatins and auristatins include, but are not limited
to, (+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.
[0227] Enediynes are a class of anti-tumor bacterial products
characterized by either nine- and ten-membered rings or the
presence of a cyclic system of conjugated triple-double-triple
bonds. Exemplary enediynes include, but are not limited to,
calicheamicin, esperamicin, and dynemicin. Calicheamicin is an
enediyne antibiotic that was originally isolated as a natural
product from the soil organism Micromonospora echinospora ssp.
calichensis (Zein et al. Science 27;240(4856):1198-1201, 1988); it
generates double-strand DNA breaks and subsequently induces
apoptosis in target cells (Zein et al. Science 27;
240(4856):1198-1201, 1988; Nicolaou et al. Chem. Biol. September;
1(1):57-66, 1994; Prokop et al. Oncogene 22:9107-9120, 2003).
[0228] In some aspects of the invention, the cytotoxic agent is an
antibiotic, such as calicheamicin, also called the LL-E33288
complex, for example, .beta.-calicheamicin, .gamma.-calicheamicin
or N-acetyl-.gamma.-calicheamicin (gamma-calicheamicin
(.gamma..sub.1)). Examples of calicheamicins suitable for use in
the present invention are disclosed, for example, in U.S. Pat. Nos.
4,671,958 4,970,198, 5,053,394, 5,037,651, 5,079,233 and 5,108,912,
which are incorporated herein by reference in its entirety. These
compounds contain a methyltrisulfide that may be reacted with
appropriate thiols to form disulfides, at the same time introducing
a functional group such as a hydrazide or other functional group
that is useful for conjugating calicheamicin to an anti-EFNA4
antibody. Disulfide analogs of calicheamicin can also be used, for
example, analogs described in U.S. Pat. Nos. 5,606,040 and
5,770,710, which are incorporated herein by reference in its
entirety. In some aspects of the invention, the disulfide analog is
N-acetyl-.gamma.-calicheamicin dimethyl hydrazide (hereinafter
"CM").
[0229] Geldanamycins are benzoquinone ansamycin antibiotic that
bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor
drugs. Exemplary geldanamycins include, but are not limited to,
17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG
(17-Dimethylaminoethylamino-17-demethoxygeldanamycin).
[0230] Maytansines or their derivatives maytansinoids inhibit cell
proliferation by inhibiting the microtubules formation during
mitosis through inhibition of polymerization of tubulin. See
Remillard et al., Science 189:1002-1005, 1975. Exemplary
maytansines and maytansinoids include, but are not limited to,
mertansine (DM1) and its derivatives as well as ansamitocin.
[0231] Taxanes are diterpenes that act as anti-tubulin agents or
mitotic inhibitors. Exemplary taxanes include, but are not limited
to, paclitaxel (e.g., TAXOL.RTM.) and docetaxel
(TAXOTERE.RTM.).
[0232] Vinca alkyloids are also anti-tubulin agents. Exemplary
vinca alkyloids include, but are not limited to, vincristine,
vinblastine, vindesine, and vinorelbine.
[0233] In some aspects of the invention, the agent is an
immunomodulating agent. Examples of an immunomodulating agent
include, but are not limited to, gancyclovier, etanercept,
tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin,
cyclophosphamide, azathioprine, mycophenolgate mofetil,
methotrextrate, glucocorticoid and its analogs, cytokines,
xanthines, stem cell growth factors, lymphotoxins, tumor necrosis
factor (TNF), hematopoietic factors, interleukins (e.g.,
interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and
IL-21), colony stimulating factors (e.g., granulocyte-colony
stimulating factor (G-CSF) and granulocyte macrophage-colony
stimulating factor (GM-CSF)), interferons (e.g.,
interferons-.alpha., -.beta. and -.gamma.), the stem cell growth
factor designated "S 1 factor," erythropoietin and thrombopoietin,
or a combination thereof.
[0234] Immunomodulatory agents useful in the invention also include
anti-hormones that block hormone action on tumors and
immunosuppressive agents that suppress cytokine production,
down-regulate self-antigen expression, or mask MHC antigens.
Representative anti-hormones include anti-estrogens including, for
example, tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapnstone, and toremifene; and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
anti-adrenal agents. Representative immunosuppressive agents
include 2-amino-6-aryl-5-substituted pyrimidines, azathioprine,
cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde,
anti-idiotypic antibodies for MHC antigens and MHC fragments,
cyclosporin A, steroids such as glucocorticosteroids, cytokine or
cytokine receptor antagonists (e.g., anti-interferon antibodies,
anti-IL10 antibodies, anti-TNF.alpha. antibodies, anti-IL2
antibodies), streptokinase, TGF.beta., rapamycin, T-cell receptor,
T-cell receptor fragments, and T cell receptor antibodies.
[0235] In some aspects of the invention, the drug is a therapeutic
protein including, but is not limited to, a toxin, a hormone, an
enzyme, and a growth factor.
[0236] Examples of a toxin protein (or polypeptide) include, but
are not limited to, dipththeria (e.g., diphtheria A chain),
Pseudomonas exotoxin and endotoxin, ricin (e.g., ricin A chain),
abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain),
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtherin toxin, Phytolaca
americana proteins (PAPI, PAPII, and PAP-S), momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,
mitogellin, restrictocin, phenomycin, enomycin, tricothecenes,
inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and
conotoxin (e.g., KIIIA or SmIIIa).
[0237] Examples of hormones include, but are not limited to,
estrogens, androgens, progestins and corticosteroids.
[0238] In some aspects of the invention, the cytotoxic agent can be
made using a liposome or biocompatible polymer. The anti-EFNA4
antibodies as described herein can be conjugated to the
biocompatible polymer to increase serum half-life and bioactivity,
and/or to extend in vivo half-lives. Examples of biocompatible
polymers include water-soluble polymer, such as polyethylene glycol
(PEG) or its derivatives thereof and zwitterion-containing
biocompatible polymers (e.g., a phosphorylcholine containing
polymer).
[0239] In some aspects of the invention, the drug is an
oligonucleotide, such as anti-sense oligonucleotides.
[0240] Additional drugs useful in the invention include
anti-angiogenic agents that inhibit blood vessel formation, for
example, farnesyltransferase inhibitors, COX-2 inhibitors, VEGF
inhibitors, bFGF inhibitors, steroid sulphatase inhibitors (e.g.,
2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE)), interleu
kin-24, thrombospondin, metallospondin proteins, class I
interferons, interleukin 12, protamine, angiostatin, laminin,
endostatin, and prolactin fragments.
[0241] Anti-proliferative agents and pro-apoptotic agents include
activators of PPAR-gamma (e.g., cyclopentenone prostaglandins
(cyPGs)), retinoids, triterpinoids (e.g., cycloartane, lupane,
ursane, oleanane, friedelane, dammarane, cucurbitacin, and limonoid
triterpenoids), inhibitors of EGF receptor (e.g., HER4),
rampamycin, CALCITRIOL.RTM. (1,25-dihydroxycholecalciferol (vitamin
D)), aromatase inhibitors (FEMARA.RTM. (letrozone)), telomerase
inhibitors, iron chelators (e.g., 3-aminopyridine-2-carboxaldehyde
thiosemicarbazone (Triapine)), apoptin (viral protein 3-VP3 from
chicken aneamia virus), inhibitors of BcI-2 and BcI-X(L),
TNF-alpha, FAS ligand, TNF-related apoptosis-inducing ligand
(TRAIL/Apo2L), activators of TNF-alpha/FAS ligand/TNF-related
apoptosis-inducing ligand (TRAIL/Apo2L) signaling, and inhibitors
of PI3K-Akt survival pathway signaling (e.g., UCN-01 and
geldanamycin).
[0242] Representative chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziidines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechiorethamine, mechiorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfarnide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, carminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic
acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,
tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such
as methotrexate and 5-fluorouracil (5-FU); folic acid analogues
such as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-EU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenal such as arninoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophospharnide glycoside; arninolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology
of Princeton, N.J.) and doxetaxel (TAXOTERE.RTM., Rhone-Poulenc
Rorer of Antony, France); chiorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aininopterin;
xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoic acid; esperamicins; and
capecitabine.
[0243] Additional therapeutic agents that may be used in accordance
with the present invention include photosensitizing agents, such as
U.S. Publication No. 20020197262 and U.S. Pat. No. 5,952,329, which
are incorporated herein by reference in its entirety, for
photodynamic therapy; magnetic particles for thermotherapy, such as
U.S. Publication No. 20030032995, which is incorporated herein by
reference in its entirety; binding agents, such as peptides,
ligands, cell adhesion ligands, etc., and prodrugs such as
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate containing prodrugs, peptide containing prodrugs,
.beta.-lactam-containing prodrugs, substituted
phenoxyacetamide-containing prodrugs or substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs that may be converted to the more active
cytotoxic free drug.
[0244] For diagnostic methods using anti-EFNA4 antibodies, a drug
may include a detectable label used to detect the presence of
EFNA4-expressing cells in vitro or in vivo. Radioisotopes that are
detectable in vivo, such as those labels that are detectable using
scintigraphy, magnetic resonance imaging, or ultrasound, may be
used in clinical diagnostic applications. Useful scintigraphic
labels include positron emitters and .gamma.-emitters.
Representative contrast agents for magnetic source imaging are
paramagnetic or superparamagnetic ions (e.g., iron, copper,
manganese, chromium, erbium, europium, dysprosium, holmium and
gadolinium), iron oxide particles, and water soluble contrast
agents. For ultrasonic detection, gases or liquids may be entrapped
in porous inorganic particles that are released as microbubble
contrast agents. For in vitro detection, useful detectable labels
include fluorophores, detectable epitopes or binding agents, and
radioactive labels.
[0245] Thus, in some aspects of the invention, the drug is an
imaging agent (e.g., a fluorophore or a PET (Positron Emission
Tomography) label, SPECT (Single-Photon Emission Computed
Tomorgraphy) label), or MRI (Magnetic Resonance Imaging) label.
[0246] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
that is detectable. Radionuclides that can serve as detectable
labels include, for example, I-131, I-123, I-125, Y-90, Re-188,
Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label might also be
a non-detectable entity such as a toxin.
[0247] Examples of fluorophores include, but are not limited to,
fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein
amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein,
erythrosine, Alexa Fluor.RTM. (e.g., Alexa 350, 405, 430, 488, 500,
514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or
750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA),
tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g.,
SR101).
[0248] Therapeutic or diagnostic radioisotopes or other labels
(e.g., PET or SPECT labels) can be incorporated in the agent for
conjugation to the anti-EFNA4 antibodies as described herein. The
isotope may be directly bound to the antibody, for example, at a
cysteine residue present in the antibody, or a chelator may be used
to mediate the binding of the antibody and the radioisotope.
Radioisotopes suitable for radiotherapy include but are not limited
to a-emitters, p-emitters, and auger electrons. For diagnostic
applications, useful radioisotopes include positron emitters and
y-emitters. An anti-EFNA4 antibody of the invention may further be
iodinated, for example, on a tyrosine residue of the antibody, to
facilitate detection or therapeutic effect of the antibody.
[0249] Examples of a radioisotope or other labels include, but are
not limited to, .sup.3H, .sup.11C, .sup.13N, .sup.14C, .sup.15N,
.sup.15O, .sup.35S, .sup.18F, .sup.32P, .sup.33P, .sup.47Sc,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.75Se, .sup.76Br, .sup.77Br,
.sup.86Y, .sup.89Zr, .sup.90Y, .sup.94Tc, .sup.95Ru, .sup.97Ru,
.sup.99Tc, .sup.103Ru, .sup.105Rh, .sup.105Ru, .sup.107Hg,
.sup.109Pd, .sup.111Ag, .sup.111In, .sup.113In, .sup.121Te,
.sup.122Te, .sup.123I, .sup.124I, .sup.125I, .sup.125Te, .sup.126I,
.sup.131I, .sup.131In, .sup.133I, .sup.142Pr, .sup.143Pr,
.sup.153Pb, .sup.153Sm, .sup.161Tb, .sup.165Tm, .sup.166Dy,
.sup.166H, .sup.167Tm, .sup.168Tm, .sup.169Yb, .sup.177Lu,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.197Pt, .sup.198Au,
.sup.199Au, .sup.201Tl, .sup.203Hg, .sup.211At, .sup.212Bi,
.sup.212Pb, .sup.213Bi, .sup.223Ra, .sup.224Ac, and .sup.225Ac.
[0250] II.D. Methods of Preparing EFNA4 Antibody-Drug
Conjugates
[0251] Also provided are methods for preparing antibody-drug
conjugates of the present invention. For example, a process for
producing an EFNA4 antibody-drug conjugate as disclosed herein can
include (a) linking the linker to the drug moiety; (b) conjugating
the linker-drug moiety to the antibody; and (c) purifying the
antibody-drug conjugate.
[0252] In one example, an antibody-drug conjugate of the formula
Ab(L-D) may be prepared by (a) adding the linker-drug moiety (e.g.
AcBut-CM) to the anti-EFNA4 antibody, or antigen-binding fragment
thereof, wherein the concentration of antibody may range from 1 to
25 mg/ml and the linker-drug moiety is at a molar ratio ranging
from about 1-15 to 1 of the anti-EFNA4 antibody; (b) incubating the
linker-drug moiety and anti-EFNA4 antibody in a non-nucleophilic,
protein-compatible, buffered solution having a pH in a range from
about 7 to 9 to produce an monomeric antibody-drug conjugate,
wherein the solution further compromises (i) a suitable organic
cosolvent, and (ii) an additive having at least one
C.sub.6-C.sub.18 carboxylic acid or its salt, and wherein the
incubation is conducted at a temperature ranging from about
0.degree. C. to about 45.degree. C., for a period of time ranging
from about 1 minute to about 24 hours; and (c) subjecting the
conjugate produced in step (b) to a chromatographic separation
process to separate antibody-drug conjugates with a DAR from 1 to
12; and provides low conjugated fraction (LCF) of below 10% from
unconjugated anti-EFNA4 antibody, linker-drug moiety, and
aggregated conjugates.
[0253] In some aspects of the invention, the linker-drug moiety is
added to the anti-EFNA4 antibody wherein the antibody has a
concentration of about 0.01 to about 1 mg/ml, or wherein the
antibody has a concentration of about 1 to about 2 mg/ml, or
wherein the antibody has a concentration of about 2 to about 3
mg/ml, or wherein the antibody has a concentration of about 3 to
about 4 mg/ml, or wherein the antibody has a concentration of about
4 to about 5 mg/ml, or wherein the antibody has a concentration of
about 5 to about 6 mg/, or wherein the antibody has a concentration
of about 6 to about 7 mg/ml, or wherein the antibody has a
concentration of about 7 to about 8 mg/ml, or wherein the antibody
has a concentration of about 8 to about 9 mg/ml, or wherein the
antibody has a concentration of about 9 to about 10 mg/ml, or
wherein the antibody has a concentration of about 10 to about 11
mg/ml, or wherein the antibody has a concentration of about 11 to
about 12 mg/ml, or wherein the antibody has a concentration of
about 12 to about 13 mg/ml, or wherein the antibody has a
concentration of about 13 to about 14 mg/ml, or wherein the
antibody has a concentration of about 14 to about 15 mg/ml, or
wherein the antibody has a concentration of about 15 to about 16
mg/ml, or wherein the antibody has a concentration of about 16 to
about 17 mg/ml, or wherein the antibody has a concentration of
about 17 to about 18 mg/ml, or wherein the antibody has a
concentration of about 18 to about 19 mg/ml, or wherein the
antibody has a concentration of about 19 to about 20 mg/ml, or
wherein the antibody has a concentration of about 20 to about 21
mg/ml, or wherein the antibody has a concentration of about 21 to
about 22 mg/ml, or wherein the antibody has a concentration of
about 22 to about 23 mg/ml, or wherein the antibody has a
concentration of about 23 to about 24 mg/ml, or wherein the
antibody has a concentration of about 24 to about 25 mg/ml, or
wherein the antibody has a concentration of about 10 mg/ml, or
wherein the antibody has a concentration of less than 10 mg/ml, As
used in the foregoing ranges of antibody, the term "about" means
+/-0.5 mg/ml.
[0254] In some aspects of the invention, the linker-drug moiety is
added to the anti-EFNA4 antibody wherein the linker-drug moiety to
anti-EFNA4 antibody is a molar ratio of 2-3 to 1, or a molar ratio
of 3-4 to 1, or a molar ratio of 4-5 to 1, or a molar ratio of 5-6
to 1, or a molar ratio of 6-7 to 1, or a molar ratio of 7-8 to 1,
or a molar ratio of 8-9 to 1, or a molar ratio of 9-10 to 1, or a
molar ratio of 10-11 to 1, or a molar ratio of 11-12 to 1, or a
molar ratio of 12-13 to 1, or a molar ratio of 13-14 to 1, or a
molar ratio of 14-15 to 1. In some aspects of the invention, the
linker-drug moiety is added to the anti-EFNA4 antibody at a molar
ratio of about 4-4.5 to 1 to thereby decrease undesirable higher
DAR antibody-drug conjugates. For example, the linker-drug moiety
is added to the anti-EFNA4 antibody at a molar ratio of 4-4.5 to 1.
Other ranges of linker-drug moiety to antibody may also be used to
reduce unconjugated antibody, low DAR and high DAR antibody-drug
conjugates.
[0255] In some aspects of the invention, the linker-drug moiety is
added to the anti-EFNA4 antibody wherein the drug is about 2% to
about 3% by weight of the anti-EFNA4 antibody, or wherein the drug
is about 3% to about 4% by weight of the anti-EFNA4 antibody, or
wherein the drug is about 4% to about 5% by weight of the
anti-EFNA4 antibody, or wherein the drug is about 5% to about 6% by
weight of the anti-EFNA4 antibody, or wherein the drug is about 6%
to about 7% by weight of the anti-EFNA4 antibody, or wherein the
drug is about 7% to about 8% by weight of the anti-EFNA4 antibody,
or wherein the drug is about 8% to about 9% by weight of the
anti-EFNA4 antibody, or wherein the drug is about 9% to about 10%
by weight of the anti-EFNA4 antibody, or wherein the drug is about
10% to about 11% by weight of the anti-EFNA4 antibody. As used in
the foregoing ranges of linker-drug moiety to antibody (5 by
weight), the term "about" means +/-0.5%.
[0256] In some aspects of the invention, the linker-drug moiety is
added to the anti-EFNA4 antibody wherein the drug is 2% to 3% by
weight of the anti-EFNA4 antibody, or wherein the drug is 3% to 4%
by weight of the anti-EFNA4 antibody, or wherein the drug is 4% to
5% by weight of the anti-EFNA4 antibody, or wherein the drug is 5%
to 6% by weight of the anti-EFNA4 antibody, or wherein the drug is
6% to 7% by weight of the anti-EFNA4 antibody, or wherein the drug
is 7% to 8% by weight of the anti-EFNA4 antibody, or wherein the
drug is 8% to 9% by weight of the anti-EFNA4 antibody, or wherein
the drug is 9% to 10% by weight of the anti-EFNA4 antibody, or
wherein the drug is 10% to 11% by weight of the anti-EFNA4
antibody.
[0257] In some aspects of the invention, the incubation described
in step (b)(ii) above is conducted at a temperature ranging from
about 0.degree. C. to about 5.degree. C., or at temperature ranging
from about 0.degree. C. to about 4.degree. C., or at temperature
ranging from about 5.degree. C. to about 10.degree. C., or at
temperature ranging from about 10.degree. C. to about 15.degree.
C., or at temperature ranging from about 15.degree. C. to about
20.degree. C., or at temperature ranging from about 20.degree. C.
to about 25.degree. C., or at temperature ranging from about
25.degree. C. to about 30.degree. C., or at temperature ranging
from about 30.degree. C. to about 35.degree. C., or at temperature
ranging from about 35.degree. C. to about 40.degree. C., or at
temperature ranging from about 40.degree. C. to about 45.degree. C.
As used in the foregoing temperature ranges, the term "about" means
+/-1.degree. C.
[0258] In some aspects of the invention, the incubation described
in step (b)(ii) above is allowed to proceed for a time sufficient
for completion of at least about 50% of the conjugation reaction,
for example, at least about 60% complete, at least about 70%
complete, at least about 80% complete, at least about 90% complete,
at least about 95% complete, or at least about 99% complete. Thus,
the reaction may be allowed to proceed for at least about 1 minute
to about 5 minutes. Longer times are also permissible so long as
aggregation of the conjugate remains at an acceptable level. In
some aspects of the invention, the reaction is mostly complete by
about 1 minute, which short duration is an improvement over prior
methods.
[0259] In some aspects of the invention, in step (c) above, the
antibody-conjugate of step (b) above is subjected to a
chromatographic separation process to select compositions, batches
and/or formulations of antibody-drug conjugates with an average DAR
in the range of about 1 to about 12, for example, an average DAR in
the range of about 2 to about 4, or an average DAR in the range of
about 3 to about 5, or an average DAR in the range of about 4 to
about 6, or an average DAR in the range of about 5 to about 7, or
an average DAR in the range of about 6 to about 8, or an average
DAR in the range of about 7 to about 9, or an average DAR in the
range of about 8 to about 10, or an average DAR in the range of
about 9 to about 11. In some aspects the compositions, batches
and/or formulations of antibody-drug conjugate may have an average
DAR of about 1, or an average DAR of about 2, an average DAR of
about 3, or an average DAR of about 4, or an average DAR of about
5, or an average DAR of about 6, or an average DAR of about 7, or
an average DAR of about 8, or an average DAR of about 9, or an
average DAR of about 10, or an average DAR of about 11. As used in
the foregoing ranges of average DAR, the term "about" means
+/-0.5%.
[0260] In some aspects the compositions, batches, and/or
formulations of antibody-drug conjugates may have an average DAR in
the range of about 3 to about 5, an average DAR in the range of
about 3 to about 4, or an average DAR in the range of about 4 to
about 5.
[0261] In some aspects the compositions, batches and/or
formulations of antibody-drug conjugate may have an average DAR in
the range of 3 to 5, or an average DAR in the range of 3 to 4, or
an average DAR in the range of 4 to 5.
[0262] In some aspects, the antibody-drug conjugate has an average
DAR of about 3.0, or an average DAR of 3.0, or an average DAR of
3.1, or an average DAR of 3.2, or an average DAR of 3.3, or an
average DAR of 3.4, or an average DAR of 3.5, or an average DAR of
3.6, or an average DAR of 3.7, or an average DAR of 3.8, or an
average DAR of 3.9. In another aspect of the invention, a
composition, batch, and/or formulation of antibody-drug conjugates
may be characterized by an average DAR of about 4.0, or an average
DAR of 4.0, or an average DAR of 4, or an average DAR of 4.1, or an
average DAR of 4.2, or an average DAR of 4.3, or an average DAR of
4.4, or an average DAR of 4.5, or an average DAR of 4.6, or an
average DAR of 4.7, or an average DAR of 4.8, or an average DAR of
4.9, or an average DAR of 5.0.
[0263] In another aspect of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by an average DAR of 12 or less, an average DAR of 11 or less, an
average DAR of 10 or less, an average DAR of 9 or less, an average
DAR of 8 or less, an average DAR of 7 or less, an average DAR of 6
or less, an average DAR of 5 or less, an average DAR of 4 or less,
an average DAR of 3 or less, an average DAR of 2 or less or an
average DAR of 1 or less.
[0264] In other aspects of the invention, a composition, batch,
and/or formulation of antibody-drug conjugates may be characterized
by an average DAR of 11.5 or less, an average DAR of 10.5 or less,
an average DAR of 9.5 or less, an average DAR of 8.5 or less, an
average DAR of 7.5 or less, an average DAR of 6.5 or less, an
average DAR of 5.5 or less, an average DAR of 4.5 or less, an
average DAR of 3.5 or less, an average DAR of 2.5 or less, an
average DAR of 1.5 or less.
[0265] In some aspects of the invention, in step (c) above, the
antibody-conjugate of step (b) above is subjected to a
chromatographic separation process to select antibody-drug
conjugates with a loading in the range of about 2% to about 10% by
weight drug, for example, loading in the range of about 2% to about
4% by weight drug, or loading in the range of about 3% to about 5%
by weight drug, or loading in the range of about 4% to about 6% by
weight drug, or loading in the range of about 5% to about 7% by
weight drug, or loading in the range of about 6% to about 8% by
weight drug, or loading in the range of about 7% to about 9% by
weight drug, or loading in the range of about 8% to about 10% by
weight drug. As used in the foregoing ranges of drug loading, the
term "about" means +/-0.5%.
[0266] In some aspects of the invention, in step (c) above, the
antibody-drug conjugate of step (b) above is subjected to a
chromatographic separation to select antibody-drug conjugates with
low conjugated fraction (LCF) of below about 10%, for example, less
than 10%, or less than about 9%, or less than 9%, or less than
about 8%, or less than 8%, or less than about 7%, or less than 7%,
or less than about 6%, or less than 6%, or less than about 5%, or
less than 5%, or less than about 4%, or less than 4%, or less than
about 3%, or less than 3%, or less than about 2%, or less than 2%,
or less than about 1%, or less than 1%, or 0%. In some aspects of
the invention, it is contemplated that an LCF above 0%, but below
10% is desirable. In the foregoing description of LCF, the term
"about" means +/-0.5% of the indicated percentages. In some aspects
of the invention, high agitation and vigorous mixing is conducted
during the addition of the linker-drug moiety, for example, as
achieved in part by addition of the linker-drug moiety into the
middle portion of the mixing vortex, which is helpful in achieving
low unconjugated fraction, which is an improvement over prior
methods.
[0267] In the context of the present invention, a monomeric
antibody-drug conjugate refers to a single antibody linked or
conjugated to any number of drug molecules without significant
aggregation of the antibodies. The percentage of antibody in a
given population having unconjugated or significantly
under-conjugated antibody is referred to as the low conjugate
fraction (LCF). The monomeric form of the conjugates as opposed to
the aggregated form has significant therapeutic value, and
minimizing the LCF and substantially reducing aggregation results
in the utilization of the antibody starting material in a
therapeutically meaningful manner by preventing the LCF from
competing with the more highly conjugated fraction (HCF).
[0268] The hydrophobic nature of many drugs, including
calicheamicins, may result in aggregation of antibody-drug
conjugates. To produce monomeric antibody-drug conjugates with
higher drug conjugates (reduced LCF) and decreased aggregation, the
conjugation reaction may be performed in a non-nucleophilic,
protein-compatible, buffered solution containing (i) ethanol as a
cosolvent and (ii) an additive having at least one C.sub.6-C.sub.18
carboxylic acid or its salt. Other protein-compatible organic
cosolvents such as ethylene glycol, propylene glycol, DMF and DMSO
may also be used. Some or all of the organic cosolvent is used to
transfer the drug into the conjugation mixture. Useful
C.sub.6-C.sub.18 carboxylic acids include decanoic acid, octanoic
acid or caprylic acid, or its salts. In one aspects of the
invention of the invention, the carboxylic acid is decanoic acid,
or the corresponding salts, such as sodium decanoate.
Representative amounts of an additive having at least one
C.sub.6-C.sub.18 carboxylic acid or its salt range from 20 mM to
100 mM, such as from 30 mM to 90 mM, or about 40 mM to 80 mM, or
about 40 mM to 50 mM. The concentration of the C.sub.6-C.sub.18
carboxylic acid or its salt may be increased to 150-300 mM and the
cosolvent dropped to 1% to 10%. Useful buffers for the preparation
of antibody-drug conjugates using N-hydroxysuccinimide (OSu) esters
or other comparably activated esters include phosphate-buffered
saline (PBS) and N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic
acid (HEPES buffer). The buffered solution used in conjugation
reactions should substantially lack free amines and nucleophiles.
As another approach, the conjugation reactions may be performed in
a non-nucleophilic, protein-compatible, buffered solution
containing t-butanol without the additional additives. See e.g.,
U.S. Pat. Nos. 5,712,374 and 5,714,586, which are incorporated
herein by reference in its entirety. Additional methods for
conjugation and calicheamicin-containing conjugates are described
in U.S. Pat. Nos. 5,739,116 and 5,877,296, which are incorporated
herein by reference in its entirety.
[0269] Optimal reaction conditions for formation of a conjugate may
be empirically determined by variation of reaction variables such
as temperature, pH, linker-calicheamicin moiety input, and additive
concentration. Conditions suitable for conjugation of other drugs
may be determined by those skilled in the art without undue
experimentation.
[0270] Other methods for preparing antibody-drug conjugates have
been described in various publications. For example, chemical
modification can be made in the antibodies either through lysine
side chain amines or through cysteine sulfhydryl groups activated
by reducing interchain disulfide bonds for the conjugation reaction
to occur. See, e.g., Tanaka et al., FEBS Letters 579:2092-2096,
2005, and Gentle et al., Bioconjugate Chem. 15:658-663, 2004.
Reactive cysteine residues engineered at specific sites of
antibodies for specific drug conjugation with defined stoichiometry
have also been described. See, e.g., Junutula et al., Nature
Biotechnology, 26:925-932, 2008. Conjugation using an acyl donor
glutamine-containing tag or an endogenous glutamine made reactive
(i.e., the ability to form a covalent bond as an acyl donor) by
polypeptide engineering in the presence of transglutaminase and an
amine (e.g., a cytotoxic agent having or attached to a reactive
amine) is also described in PCT International Publication No. WO
2012/059882.
[0271] To further increase the number of drug molecules per
antibody-drug conjugate, the drug may be conjugated to polyethylene
glycol (PEG), including straight or branched polyethylene glycol
polymers and monomers. A PEG monomer is of the formula:
(CH.sub.2CH.sub.2O)--. Drugs and/or peptide analogs may be bound to
PEG directly or indirectly, i.e. through appropriate spacer groups
such as sugars. A PEG-antibody-drug composition may also include
additional lipophilic and/or hydrophilic moieties to facilitate
drug stability and delivery to a target site in vivo.
Representative methods for preparing PEG-containing compositions
may be found in U.S. Pat. Nos. 6,461,603; 6,309,633; and 5,648,095,
among other places.
[0272] For example, to increase the amount of calicheamicin in
antibody-calicheamicin conjugates, the antibody is conjugated to
PEG prior to conjugation with calicheamicin, for example, using
PEG-SPA, PEG-SBA, or PEG-bis-maleimide. Antibody-drug conjugates
prepared using PEG may show reduced binding affinity for the target
antigen, but are still effective as a result of increased drug
load.
[0273] Following conjugation, the chromatographic separation of
step (c) above, the conjugates may be separated from unconjugated
reactants and/or aggregated forms of the conjugates by conventional
methods. This can include processes such as size exclusion
chromatography (SEC), ultrafiltration/diafiltration, ion exchange
chromatography (IEC), chromatofocusing (CF) HPLC, FPLC, or
Sephacryl S-200 chromatography. The chromatographic separation may
also be accomplished by hydrophobic interaction chromatography
(HIC), which offers some advantages over SEC including (1) a
capability to efficiently reduce LCF content as well as aggregate;
(2) accommodation of large reaction volumes; and (3) minimal
dilution of the product. Suitable HIC media includes Phenyl
Sepharose 6 Fast Flow chromatographic medium, Butyl Sepharose 4
Fast Flow chromatographic medium, Octyl Sepharose 4 Fast Flow
chromatographic medium, Toyopearl Ether-650M chromatographic
medium, Macro-Prep methyl HlC medium or Macro-Prep t-Butyl HIC
medium.
[0274] In some aspects of the invention, the chromatographic
separation is performed using Butyl Sepharose 4 Fast Flow
chromatographic medium. When using the customized gradient as
described in Example 6, higher DAR species that remain bound to the
column are removed, which is an improvement over prior methods.
[0275] In some aspects the purification process, may include a
centrifuge cell removal step, optionally a Protein A affinity
capture step followed by one or two orthogonal chromatographic
polishing steps, a virus filtration step, and a tangential flow
filtration step for concentration and formulation.
[0276] A typical anti-EFNA4-ActBut-CM antibody-drug conjugate
preparation contains predominantly (.about.95%) conjugated antibody
containing 3-5 moles CM per mole antibody, for example, 3-4 moles
CM per antibody, or 4-5 moles CM per antibody. In in vivo studies,
EFNA4 antibody-drug conjugates prepared with this molar range for
drug loading are highly efficacious with minimal toxicity.
[0277] The EFNA4-ActBut-CM antibody-drug conjugate has been
reproducibly prepared at the laboratory scale (10-200 mg). DAR or
drug loading, which is expressed as pg CM/mg monoclonal antibody,
is determined by dividing the CM concentration (.mu.g/mL) by the
antibody concentration (mg/mL). These values are determined by
measuring the UV absorbance of the conjugate solution at 280 nm
(antibody) and 310 nm (calicheamicin). It is important to note that
this is an average drug loading and that the actual drug loading is
a quasi-gaussian distribution centered on the average drug loading
value, i.e., some of the antibody is loaded higher than average and
some of the antibody is loaded lower than the average. As compared
to known antibody-calicheamicin conjugates (e.g., CMC-676 and
CMC-544), the DAR distribution is very narrow, and 3 to 5 DAR
species (which are the most desired) make up .about.75% of the
total. Unconjugated antibody (low conjugated fraction or LCF) can
be measured using analytical HIC-HPLC (hydrophobic interaction
high-performance liquid chromatography). This value is a measure of
CM distribution on the antibody and does not generally affect the
amount of CM dosed. Unconjugated CM, which can be measured using
ELISA, refers to the amount of CM that is not conjugated to the
antibody and is expressed in terms of percent of total CM.
Drug-loading assays do not differentiate between unconjugated and
conjugated CM. The amount of unconjugated CM is undetectable or
negligible when using drug-loading assays, and therefore these
assays effectively measure the amount of conjugated CM.
[0278] Analytical methods can be used to assay for release and
stability testing of humanized EFNA4-AcBut-CM antibody-drug
conjugates. The conjugates can be evaluated for identity (IEF),
strength (total protein and total CM loading), purity (unconjugated
CM, low conjugated antibody, aggregate content and SDS-PAGE
Reduced), and immunoaffinity (antigen binding ELISA). Additional
assays known to those of skill in the art can be used. Using these
assays, batch-to-batch consistency can be maintained in commercial
manufacture.
III. Functional Assays for Characterization of EFNA Antibody-Drug
Conjugates
[0279] The present invention further discloses in vitro and in vivo
assays to characterize activities of an EFNA4 antibody-drug
conjugate, including EFNA binding activity, cellular
internalization following binding to EFNA4 antigen presented on a
cell surface, and targeting to EFNA4-expressing cells in a subject.
In some aspects of the invention, EFNA4 antibody-drug conjugates
are characterized by the neutralizing or depleting aspects of the
antibody, or antigen-binding fragment thereof. In some aspects of
the invention, EFNA4 antibody-drug conjugates are characterized by
unexpected efficacy of a particular drug as compared to lack of
efficacy of an alternate drug. In some aspects of the invention,
EFNA4 antibody-drug conjugates are characterized as outperforming a
standard-of-care therapeutic agent having a same mode of action as
the drug.
[0280] Techniques for detecting binding of EFNA4 antibody-drug
conjugates to an EFNA4 antigen, or other EFNA antigen, are known in
the art, including for example, BIACORE.RTM. assays. Additional
representative techniques include centrifugation, affinity
chromatography and other immunochemical methods. See e.g., Manson
(1992) Immunochemical Protocols, Humana Press, Totowa, N.J., United
States of America; Ishikawa (1999) Ultrasensitive and Rapid Enzyme
Immunoassay, Elsevier, Amsterdam/New York. Antigen binding assays
may be performed using isolated EFNA4 antigen or EFNA4-expressing
cells.
[0281] The binding specificity of EFNA4 antibody-drug conjugates
may be further described by definition of a binding epitope, i.e.,
identification of residues, including nonadjacent residues that
participate in antigen binding, and/or definition of residues that
influence antigen binding.
[0282] Internalization of EFNA4 antibody-drug conjugates by
EFNA-expressing cells may be assayed by observing the amount of
antibodies or conjugates bound to the surface of the
EFNA-expressing cells over time. Selected EFNA ligands or their
isoforms may be present in a soluble form, and at least some EFNA4
likely remains associated with the cell surface thereby allowing
for internalization of the antibodies disclosed herein.
Accordingly, anti-EFNA4 antibody-drug conjugates of the present
invention may be internalized by cells that express EFNA4. For
example, an anti-EFNA4 antibody-drug conjugates that binds to EFNA4
on the surface of a tumor initiating cell may be internalized by
the tumor initiating cell.
[0283] Internalization of EFNA4 antibodies may be assessed using a
functional assay in which cells are incubated with the EFNA4
antibody and a secondary antibody Fab fragment that is conjugated
to the saporin toxin. Cell viability is then measured by any
suitable method, with cellular cytotoxicity indicative of antibody
internalization. See Example 5.
[0284] In some aspects of the invention, the antibody, or
antigen-binding fragment thereof, of the disclosed EFNA4
antibody-drug conjugates is an "antagonist" as used in the broadest
sense, i.e., any molecule that partially or fully blocks, inhibits,
or neutralizes a biological activity of a native target disclosed
herein or the transcription or translation thereof. The terms
"inhibit" or "neutralize" as used herein with respect to
bioactivity of an antibody of the invention mean the ability of the
antibody to substantially antagonize, prohibit, prevent, restrain,
slow, disrupt, eliminate, stop, reduce or reverse e.g. progression
or severity of that which is being inhibited including, but not
limited to, a biological activity. For example, in some aspects of
the invention, anti-EFNA4 antibody-drug conjugate facilitate cell
killing upon internalization of the antibody-drug conjugate.
[0285] More particularly, the term neutralizing antibody or
neutralizing antagonist refers to an antibody or antagonist that
binds to or interacts with an EFNA ligand and prevents binding or
association of the ligand to its binding partner (e.g., EPHA
receptor) thereby interrupting the biological response that
otherwise would result from the interaction of the molecules. For
EFNA4, a neutralizing antibody or antagonist will preferably
diminish the ability of EFNA4 to bind to EPHA4 by at least about
20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more.
It will be appreciated that this diminished activity may be
measured directly using art recognized techniques or may be
measured by the impact such reduction will have on EPHA4 receptor
activity.
[0286] In other aspects of the invention the anti-EFNA4
antibody-drug conjugates of the present invention may be depleting
antibodies. The term depleting antibody refers to an antibody that
binds to or associates with EFNA4 on or near the cell surface and
induces, promotes or causes the death or elimination of the cell
(e.g., by complement-dependent cytotoxicity or antibody-dependent
cellular cytotoxicity). Preferably a depleting antibody will be
able to remove, eliminate or kill at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 90%, 95%, 97%, or 99% of tumor perpetuating cells in
a defined cell population.
[0287] Functional assays also include methods for assessing
anti-cancer activity of antibody-drug conjugates, for example, an
ability to destroy existing cancer cells, or to delay or prevent
growth of cancer cells. Cancers targeted by antibody-drug
conjugates of the invention include both primary and metastasized
tumors and carcinomas of any tissue in a subject, including
carcinomas and hematopoietic malignancies such as leukemias and
lymphomas.
[0288] EFNA4 antibody-drug conjugates having growth inhibitory
activity can eliminate EFNA-expressing cells or to prevent or
reduce proliferation of EFNA-expressing cancer cells.
Representative methods for rapid in vitro assessment of cell growth
inhibition are described in Jones et al. (2001) J. Immunol. Methods
254:85-98.
[0289] EFNA4 antibody-drug conjugates may also include an ability
to induce cell death, for example, programmed cell death
characterized by nuclear DNA degradation, nuclear degeneration and
condensation, loss of membrane integrity, and phagocytosis.
Representative assays to assess cell are described in Hoves et al.
(2003) Methods 31:127-34; Peng et al. (2002) Chin. Med. Sci. J.
17:17-21; Yasuhara et al. (2003) J. Histochem. Cytochem.
51:873-885.
[0290] For example, to assess the cytotoxicity of EFNA4
antibody-drug conjugates in vitro, EFNA4-expressing cancer cells
and control cells are cultured in the presence EFNA4 antibody-drug
conjugates and separately with free drug. The cytotoxicity of each
agent is reported as ED50 (ng/ml), which is the amount of drug
given as conjugate or as free drug that causes 50% reduction of a
cell culture relative to an untreated control. The number of cells
in culture is determined using a vital dye (MTS) following drug
exposure.
[0291] To assess the cytotoxicity of EFNA4 antibody-drug conjugates
in vivo, tumors are prepared in NOD/SCID mice by subcutaneous
injection of various cancer cells. EFNA4 antibody-drug conjugates
and control compounds may be administered to tumor-bearing mice,
for example, by intraperitoneal injection twice a week for two
weeks (q4dx4). Measurable therapeutic outcomes include inhibition
of tumor cell growth. See Example 8.
[0292] Further, the present invention provides for EFNA4
antibody-drug conjugates that may deplete, silence, neutralize,
eliminate or inhibit growth, propagation or survival of tumor
cells, including tumor initiating cells (TIC), and/or associated
neoplasia through a variety of mechanisms, including agonizing or
antagonizing selected pathways or eliminating specific cells
depending, for example, on the anti-EFNA4 antibody, or dosing and
method of delivery. See Example 9.
[0293] As used herein, the term tumor initiating cell (TIC)
encompasses both tumor perpetuating cells (TPC; i.e., cancer stem
cells or CSC) and highly proliferative tumor progenitor cells
(TProg), which together generally include a unique subpopulation
(i.e. 0.1-40%) of a bulk tumor or mass. For the purposes of the
instant disclosure the terms tumor perpetuating cells and cancer
stem cells are equivalent and may be used interchangeably herein.
Conversely, TPC differ from TProg in that they can completely
recapitulate the composition of tumor cells existing within a tumor
and have unlimited self-renewal capacity as demonstrated by serial
transplantation (two or more passages through mice) of low numbers
of isolated cells. As used herein, the term "tumor initiating cell"
also refers to cancer stem cells of various hematologic
malignancies, which are not characterized by a tumor per se.
[0294] The present invention provides EFNA4 antibody-drug
conjugates that target tumor initiating cells (TIC), and especially
tumor perpetuating cells (TPC), thereby facilitating the treatment,
management or prevention of neoplastic disorders and
hyperproliferative disorders. More specifically, specific tumor
cell subpopulations express EFNA4 and likely modify localized
coordination of morphogen signaling important to cancer stem cell
self-renewal and cell survival. Thus, EFNA4 antibody-drug
conjugates may be used to reduce the frequency of TICs upon
administration to a subject. The reduction in tumor initiating cell
frequency may occur as a result of a) elimination, depletion,
sensitization, silencing or inhibition of tumor initiating cells;
b) controlling the growth, expansion or recurrence of tumor
initiating cells; c) interrupting the initiation, propagation,
maintenance, or proliferation of tumor initiating cells; or d) by
otherwise hindering the survival, regeneration and/or metastasis of
the tumorigenic cells. In some aspects of the invention, the
reduction in the frequency of tumor initiating cells occurs as a
result of a change in one or more physiological pathways. The
change in the pathway, whether by reduction or elimination of the
tumor initiating cells or by modifying their potential (e.g.,
induced differentiation, niche disruption) or otherwise interfering
with their ability to exert effects on the tumor environment or
other cells, in turn allows for the more effective treatment of
EFNA4-associated disorders by inhibiting tumorigenesis, tumor
maintenance and/or metastasis and recurrence.
[0295] Among the methods that can be used to assess such a
reduction in the frequency of tumor initiating cells is limiting
dilution analysis either in vitro or in vivo, preferably followed
by enumeration using Poisson distribution statistics or assessing
the frequency of predefined definitive events such as the ability
to generate tumors in vivo or not. It is also possible to determine
reduction of frequency values through well-known flow cytometric or
immunohistochemical means. As to all the aforementioned methods
see, for example, Dylla et al., PLoS ONE 3(6):e2428, 20082 &
Hoey et al, Cell Stem Cell 5:168-177, 2009, each of which is
incorporated herein by reference in its entirety. Other methods
compatible with the instant invention that may be used to calculate
tumor initiating cell frequency, include quantifiable flow
cytometric techniques and immunohistochemical staining procedures.
Representative methods are described in Example 9.
[0296] Using any of the above-referenced methods it is then
possible to quantify the reduction in frequency of TIC (or the TPC
therein) provided by the disclosed EFNA4 antibody-drug conjugates
in accordance with the teachings herein. In some instances, the
EFNA4 antibody-drug conjugates of the instant invention may reduce
the frequency of TIC (by a variety of mechanisms noted above,
including elimination, induced differentiation, niche disruption,
silencing, etc.) by 10%, 15%, 20%, 25%, 30% or even by 35%. In
other aspects of the invention, the reduction in frequency of TIC
may be on the order of 40%, 45%, 50%, 55%, 60% or 65%. In certain
aspects of the invention, the disclosed compounds my reduce the
frequency of TIC by 70%, 75%, 80%, 85%, 90% or even 95%. It will be
appreciated that any reduction of the frequency of the TIC likely
results in a corresponding reduction in the tumorigenicity,
persistence, recurrence and aggressiveness of the neoplasia.
[0297] Amassing evidence supports the hypothesis that tumor growth,
resistance to therapy, and disorder relapse are controlled by TPCs.
The frequency of TPC may vary in a tumor type or between patients
with the same tumor type as a product of disorder stage and/or
degree of differentiation within the tumor. TPC can be identified
and enriched using panels of cell surface markers that often
overlap in their expression among patients with certain types of
cancer. TPC are best defined by their functional ability to
initiate tumors upon serial transplantation, whereas
non-tumorigenic (NTG) cells are devoid of this capacity. Solid
tumor cells enriched for their unique tumor initiating capacity
were first identified in breast cancer; however, breast cancer
includes a spectrum of malignancies. To date, the scientific
community has failed to associate specific TPC identities with
particular disorder subtypes, which may underlie discrepant results
both across and within groups and may also increase the likelihood
of failed translation to the clinic.
[0298] The present invention provides a combination of new cell
surface makers that improve the enrichment of TPCs. In particular
aspect, the invention provides a combination of new cell surface
makers that facilitate the enrichment of triple negative breast
cancer (TNBC) TPCs. The present invention further provides for the
identification of EFNA4 as a novel TPC-associated therapeutic
target in TNBC; the expression level of which is significantly
higher than in other breast cancer subtypes and normal tissue.
[0299] The pharmacokinetics of EFNA4 antibody-drug conjugates can
be evaluated and compared to the pharmacokinetics of unconjugated
calicheamicin in various animals. For example, this can be done
following a single intravenous bolus administration in female
NOD/SCID mice, male Sprague-Dawley rats, and female cynomolgus
monkeys. Pharmacokinetics of EFNA4 antibody-drug conjugates are
generally characterized by low clearance, low volume of
distribution, and long apparent terminal half-life in various
species. The serum concentrations of unconjugated calicheamicin
derivatives are expected to be below the quantification limit. The
toxicity profile for these conjugates in single-dose toxicity
ranging studies is expected to be similar to that obtained for
other antibody-calicheamicin conjugates at comparable doses.
[0300] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is a tumor cell, e.g.,
breast, ovarian, colorectal, liver and lung. Various methods are
available for evaluating the cellular events associated with
apoptosis. For example, phosphatidyl serine (PS) translocation can
be measured by annexin binding; DNA fragmentation can be evaluated
through DNA laddering; and nuclear/chromatin condensation along
with DNA fragmentation can be evaluated by any increase in
hypodiploid cells.
[0301] As used herein "antibody-dependent cell-mediated
cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.
natural killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and NK cells. Alternatively, or additionally, ADCC activity
of the molecule of interest may be assessed in vivo, e.g., in an
animal model such as that disclosed in Clynes et al., PNAS (USA),
95:652-656 (1998).
[0302] "Complement dependent cytotoxicity" or "CDC" refers to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g. an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202: 163-171 (1997), may be
performed.
[0303] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. The cells may express
Fc.gamma.RIII and carry out antigen-dependent cell-mediated
cyotoxicity (ADCC) effector function. Examples of human leukocytes
that mediate ADCC include but are not limited to peripheral blood
mononuclear cells (PBMC), natural killer (NK) cells, monocytes,
macrophages, eosinophils, and neutrophils, with PBMCs and NK cells
being preferred. Antibodies that have ADCC activity are typically
of the IgG1 or IgG3 isotype. Such ADCC-mediating antibodies can
also be made by engineering a variable region from a non-ADCC
antibody or variable region fragment to an IgG1 or IgG3 isotype
constant region.
IV. Uses of EFNA Antibody-Drug Conjugates
[0304] The antibodies and the antibody drug-conjugates of the
present invention are useful in various applications including, but
are not limited to, therapeutic treatment methods and diagnostic
treatment methods.
[0305] IV.A. In Vitro Applications
[0306] The present invention provides in vitro methods using EFNA4
antibody-drug conjugates. For example, the disclosed antibodies may
be used, either alone or in combination with cytotoxic agents or
other drugs to specifically bind EFNA-positive cancer cells to
deplete such cells from a cell sample. Methods are also provided
for inducing apoptosis and/or inhibition of cell proliferation via
contacting EFNA-expressing cells with an EFNA4 antibody-drug
conjugate. Representative in vitro methods are described herein
above under the heading of "Functional Assays for Characterization
of EFNA4 antibody-drug conjugates."
[0307] EFNA4 antibody-drug conjugates of the invention also have
utility in the detection of EFNA-positive cells in vitro based on
their ability to specifically bind EFNA4 antigen. A method for
detecting EFNA-expressing cells may include: (a) preparing a
biological sample having cells; (b) contacting an EFNA4
antibody-drug conjugates with the biological sample in vitro,
wherein the drug is a detectable label; and (c) detecting binding
the EFNA4 antibody-drug conjugates.
[0308] EFNA4 antibody-drug conjugates disclosed herein are also
useful for reducing the frequency of tumor initiating cells in a
tumor sample. For example, the method can include the steps
contacting in vitro a tumor cell population, wherein the population
comprises tumor initiating cells and tumor cells other than tumor
initiating cells, with an EFNA4 antibody-drug conjugate; whereby
the percentage of tumor initiating cells in the cell population is
reduced. As used herein, the term "tumor initiating cell" also
refers to cancer stem cells of various hematologic malignancies,
which are not characterized by a tumor per se. Representative tumor
samples include any biological or clinical sample which contains
tumor cells, for example, a tissue sample, a biopsy, a blood
sample, plasma, saliva, urine, seminal fluid, etc. Representative
methods are described in Example 9.
[0309] IV.B. Therapeutic Applications
[0310] EFNA4 associated disorders include but are not limited to as
breast cancer, such as triple-negative breast cancer (TNBC);
ovarian cancer; colorectal cancer; leukemias, such as chronic
lymphocytic leukemia (CLL); liver cancer, such as hepatocellular
carcinoma (HCC);and lung cancer, such as non-small cell lung cancer
(NSCLC) and small cell lung cancer (SCLC).
[0311] The phrase "effective amount", "effective dosage" or as used
herein refers to an amount of a drug, compound or pharmaceutical
composition necessary to achieve any one or more beneficial or
desired therapeutic results. For prophylactic use, beneficial or
desired results include eliminating or reducing the risk, lessening
the severity, or delaying the outset of the disorder, including
biochemical, histological and/or behavioral symptoms of the
disorder, its complications and intermediate pathological
phenotypes presenting during development of the disorder. For
therapeutic use, beneficial or desired results include clinical
results such as reducing incidence or amelioration of one or more
symptoms of various EFNA4 associated disorders decreasing the dose
of other medications required to treat the disorder, enhancing the
effect of another medication, and/or delaying the progression of
the EFNA4 associated disorder of patients.
[0312] In one aspect, the invention provides a method for treating
a disorder associated with EFNA4 expression in a subject. The
invention also provides an antibody-drug conjugate, or a
pharmaceutical composition, as described herein, for use in a
method for treating a disorder associated with EFNA4 expression in
a subject. The invention further provides the use of an
antibody-drug conjugate, or a pharmaceutical composition, as
described herein, in the manufacture of a medicament for treating a
disorder associated with EFNA4 expression in a subject.
[0313] In some aspects of the invention, the method of treating a
disorder associated with EFNA4 expression in a subject includes
administering to the subject in need thereof an effective amount of
a composition (e.g., pharmaceutical composition) having the EFNA4
antibody-drug conjugates as described herein. The disorders
associated with EFNA4 expression include, but are not limited to,
abnormal EFNA4 expression, altered or aberrant EFNA4 expression,
EFNA4 overexpression, and a proliferative disorder (e.g.,
cancer).
[0314] In one aspect of the invention, the disorder is cancer,
including, but not limited to, mesothelioma, hepatobilliary
(hepatic and billiary duct), hepatocellular carcinoma, a primary or
secondary CNS tumor, a primary or secondary brain tumor, lung
cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin
cancer, cancer of the head or neck, melanoma, ovarian cancer, colon
cancer, rectal cancer, cancer of the anal region, stomach cancer,
gastrointestinal (gastric, colorectal, and duodenal), breast
cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma
of the endometrium, carcinoma of the cervix, carcinoma of the
vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the
esophagus, cancer of the small intestine, cancer of the endocrine
system, cancer of the thyroid gland, cancer of the parathyroid
gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer
of the urethra, cancer of the penis, prostate cancer, testicular
cancer, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, cancer of the bladder, cancer of the kidney
or ureter, renal cell carcinoma, carcinoma of the renal pelvis,
neoplasms of the central nervous system (CNS), primary CNS
lymphoma, non hodgkins's lymphoma, spinal axis tumors, brain stem
glioma, pituitary adenoma, adrenocortical cancer, gall bladder
cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, retinoblastoma, or a combination of one or more of
the cancers disclosed herein.
[0315] In a particular aspect of the invention, cancers suitable
for targeting using anti-EFNA4 antibody-drug conjugates include
EFNA4-expressing primary and metastatic cancers, such as breast
cancer, including triple-negative breast cancer (TNBC); ovarian
cancer; colorectal cancer; leukemias, such as chronic lymphocytic
leukemia (CLL); liver cancer, such as hepatocellular carcinoma
(HCC); and lung cancer, such as non-small cell lung cancer (NSCLC)
and small cell lung cancer (SCLC). In some aspects of the
invention, provided is a method of inhibiting tumor growth or
progression in a subject who has a EFNA4 expressing tumor,
including administering to the subject in need thereof an effective
amount of a composition having the EFNA4 antibody-drug conjugates
as described herein. In other aspects of the invention, provided is
a method of inhibiting metastasis of EFNA4 expressing cancer cells
in a subject, including administering to the subject in need
thereof an effective amount of a composition having the EFNA4
antibody-drug conjugates as described herein. In other aspects of
the invention, provided is a method of inducing regression of a
EFNA4 expressing tumor regression in a subject, including
administering to the subject in need thereof an effective amount of
a composition having the EFNA4 antibody-drug conjugates as
described herein. In other aspects, the invention provides an
antibody-drug conjugate, or a pharmaceutical composition, as
described herein, for use in a method as described above. In other
aspects the invention provides the use of an antibody-drug
conjugate, or a pharmaceutical composition, as described herein, in
the manufacture of a medicament for use in the methods described
above.
[0316] Thus, patients to be treated with EFNA4 antibody-drug
conjugates of the invention may be selected based on biomarker
expression, including but not limited to mRNA (qPCR) of bulk tumor
samples and elevated expression of EFNA4 antigen which results in a
patient population selected for enriched target expression rather
than tumor origin or histology. Target expression can be measured
as a function of the number of cells staining combined with the
intensity of the cells staining. For example, classification of
high expression of EFNA4 includes those patients with greater than
30% (i.e., 40%, 50% or 60%) of the cells tested by
immunohistochemical staining positive for EFNA4 at a level of 3+
(on a scale of 1 to 4), while moderate expression of the EFNA4 can
include those patients with greater than 20% of the cell cells
staining at 1+ to 2+. Target expression can also be measured by
detecting EFNA expression on tumor initiating cells (TIC) as
described herein.
[0317] The expression of EFNA4 in TNBC is specific as it is higher
than in other breast cancer subtypes and normal tissues. In
general, tumors with lower expression levels of EFNA4 exhibited the
Claudin-low TNBC molecular signature, which suggests the potential
translational relevance of that classification for patient
selection strategies. The present invention provides for selecting
patients having an increased expression of EFNA4 and treating the
patients with an EFNA4 ADC disclosed herein. Further, copy number
gain of EFNA4 and mRNA levels in breast cancer, ovarian cancer and
hepatocellular carcinoma may be relevant to determine genetic basis
for overexpression of EFNA4 and used in patient selection
strategies. The present invention further provides for selecting
patients having an increase in copy number of EFNA4 and treating
the patients with an EFNA4 ADC disclosed herein. The present
invention further provides for selecting patients having an
increase in mRNA levels of EFNA4 and treating the patients with an
EFNA4 ADC disclosed herein.
[0318] Biomarkers other than expression of EFNA4 can be also used
for patient selection, including characterization of the tumor
based on multi-drug resistance (MDR), for example. Nearly 50% of
human cancers are either completely resistant to chemotherapy or
respond only transiently, after which they are no longer affected
by commonly used anticancer drugs. This phenomenon is referred to
as MDR and is inherently expressed by some tumor types, while
others acquire MDR after exposure to chemotherapy treatment. The
drug efflux pump P-glycoprotein mediates a majority of the MDR
associated with cytotoxic chemotherapeutics. Phenotypic and
functional analysis of MDR mechanisms present in cancer patient
tumor specimens can be conducted in order to relate specific MDR
mechanism(s) with resistance to chemotherapy in specific tumor
types.
[0319] The present invention provides that TNBC TPC are
significantly enriched among ESA.sup.+CD46.sup.+CD324.sup.+ cells,
but not in the CD324.sup.- counterparts. The present invention
further provides the use of ESA and/or CD46 and/or CD324 as markers
for selecting patients and treating the patients with an EFNA4 ADC
disclosed herein. The present invention further provides the use of
ESA and/or CD46 and/or CD324 as markers for selecting TNBC patients
and treating the patients with an EFNA4 ADC disclosed herein.
[0320] Cancer growth or abnormal proliferation refers to any one of
a number of indices that suggest change within cells to a more
developed cancer form or disorder state. Inhibition of growth of
cancer cells or cells of a non-neoplastic proliferative disorder
may be assayed by methods known in the art, such as delayed tumor
growth and inhibition of metastasis. Other indices for measuring
inhibition of cancer growth include a decrease in cancer cell
survival, a decrease in tumor volume or morphology (for example, as
determined using computed tomographic (CT), sonography, or other
imaging method), destruction of tumor vasculature, improved
performance in delayed hypersensitivity skin test, an increase in
the activity of cytolytic T-lymphocytes, and a decrease in levels
of tumor-specific antigens.
[0321] Desired outcomes of the disclosed therapeutic methods are
generally quantifiable measures as compared to a control or
baseline measurement. As used herein, relative terms such as
"improve," "increase," or "reduce" indicate values relative to a
control, such as a measurement in the same individual prior to
initiation of treatment described herein, or a measurement in a
control individual (or multiple control individuals) in the absence
of the treatment described herein. A representative control
individual is an individual afflicted with the same form of
hyperproliferative disorder as the individual being treated, who is
about the same age as the individual being treated (to ensure that
the stages of the disorder in the treated individual and the
control individual are comparable.
[0322] Changes or improvements in response to therapy are generally
statistically significant. As used herein, the term "significance"
or "significant" relates to a statistical analysis of the
probability that there is a non-random association between two or
more entities. To determine whether or not a relationship is
"significant" or has "significance," statistical manipulations of
the data can be "p-value." Those p-values that fall below a
user-defined cut-off point are regarded as significant. A p-value
less than or equal to 0.1, less than 0.05, less than 0.01, less
than 0.005, or less than 0.001 may be regarded as significant.
[0323] As described herein above under the heading "III. Functional
Assays for Characterization of EFNA Antibody-Drug Conjugates," the
present invention also provides methods for targeting tumor
initiating cells. More particularly, EFNA4 antibody-drug conjugates
of the invention may deplete, silence, neutralize, eliminate or
inhibit growth, propagation or survival of tumor cells, including
tumor initiating cells.
[0324] Thus, EFNA4 antibody-drug conjugates disclosed herein are
also useful for reducing the frequency of tumor initiating cells in
a tumor sample. For example, the method can include the steps
contacting a tumor cell population, wherein the population
comprises tumor initiating cells and tumor cells other than tumor
initiating cells, with an EFNA4 antibody-drug conjugate; whereby
the percentage of tumor initiating cells in the cell population is
reduced. As used herein, the term "tumor initiating cell" also
refers to cancer stem cells of various hematologic malignancies,
which are not characterized by a tumor per se. The contacting step
may be performed in vitro, wherein the tumor cell population is
contained in a biological sample, as described herein above.
Alternatively, the contacting step may be performed in vivo as
occurs following administration of an EFNA4 antibody-drug conjugate
to a subject.
[0325] IV.C. In Vivo Detection and Diagnosis
[0326] In another aspect, provided is a method of detecting,
diagnosing, and/or monitoring a disorder associated with EFNA4
expression. For example, the EFNA4 antibodies as described herein
can be labeled with a detectable moiety such as an imaging agent
and an enzyme-substrate label. The antibodies as described herein
can also be used for in vivo diagnostic assays, such as in vivo
imaging (e.g., PET or SPECT), or a staining reagent.
[0327] Following administration of an EFNA4 antibody-drug conjugate
to a subject, wherein the drug is a detectable label, and after a
time sufficient for binding, the biodistribution of
EFNA4-expressing cells bound by the antibody may be visualized. The
disclosed diagnostic methods may be used in combination with
treatment methods. In addition, EFNA4 antibody-drug conjugates of
the invention may be administered for the dual purpose of detection
and therapy.
[0328] Representative non-invasive detection methods include
scintigraphy (e.g., SPECT (Single Photon Emission Computed
Tomography), PET (Positron Emission Tomography), gamma camera
imaging, and rectilinear scanning), magnetic resonance imaging
(e.g., convention magnetic resonance imaging, magnetization
transfer imaging (MTI), proton magnetic resonance spectroscopy
(MRS), diffusion-weighted imaging (DWI) and functional MR imaging
(fMRI)), and ultrasound.
[0329] IV.D. Formulation
[0330] The present invention further provides pharmaceutical
compositions including any of the EFNA4 antibody-drug conjugates
disclosed herein and a pharmaceutically acceptable carrier.
Further, the compositions can include more than one EFNA4 antibody
or EFNA4 antibody-drug conjugate (e.g., a mixture of EFNA4
antibodies that recognize different epitopes of EFNA4). Other
exemplary compositions include more than one EFNA4 antibody or
EFNA4 antibody-drug conjugate that recognize the same epitope(s),
or different species of EFNA4 antibodies or EFNA4 antibody-drug
conjugate that bind to different epitopes of EFNA4 (e.g., human
EFNA4).
[0331] The composition used in the present invention can further
include pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 21st
Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
"Pharmaceutically acceptable salt" as used herein refers to
pharmaceutically acceptable organic or inorganic salts of a
molecule or macromolecule. Pharmaceutically acceptable excipients
are further described herein.
[0332] Various formulations of the EFNA4 antibody or the EFNA4
antibody-drug conjugate may be used for administration. In some
aspects of the invention, the EFNA4 antibody or the EFNA4
antibody-drug conjugate may be administered neat. The EFNA4
antibody or the EFNA4 antibody-drug conjugate and a
pharmaceutically acceptable excipient may be in various
formulations. Pharmaceutically acceptable excipients are known in
the art, and are relatively inert substances that facilitate
administration of a pharmacologically effective substance. For
example, an excipient can give form or consistency, or act as a
diluent. Suitable excipients include but are not limited to
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy 20th Ed. Mack
Publishing, 2000.
[0333] In some aspects of the invention, these agents are
formulated for administration by injection (e.g.,
intraperitoneally, intravenously, subcutaneously, intramuscularly,
etc.). Accordingly, these agents can be combined with
pharmaceutically acceptable vehicles such as saline, Ringer's
solution, dextrose solution, and the like. The particular dosage
regimen, i.e., dose, timing and repetition, will depend on the
particular individual and that individual's medical history.
[0334] Therapeutic formulations of the EFNA4 antibody or the EFNA4
antibody-drug conjugate used in accordance with the present
invention are prepared for storage by mixing an antibody having the
desired degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington, The Science and
Practice of Pharmacy 21st Ed. Mack Publishing, 2005), in the form
of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and may include buffers
such as phosphate, citrate, and other organic acids; salts such as
sodium chloride; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0335] Liposomes containing the EFNA4 antibody or the EFNA4
antibody-drug conjugate are prepared by methods known in the art,
such as described in Eppstein, et al., Proc. Natl. Acad. Sci. USA
82:3688-3692, 1985; Hwang, et al., Proc. Natl Acad. Sci. USA
77:4030-4034, 1980; and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556. Particularly useful liposomes can be generated by
the reverse phase evaporation method with a lipid composition
including phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0336] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy 21st Ed. Mack Publishing, 2005.
[0337] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or `poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0338] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic EFNA4
antibody or EFNA4 antibody-drug conjugate compositions are
generally placed into a container having a sterile access port, for
example, an intravenous solution bag or vial having a stopper
pierceable by a hypodermic injection needle.
[0339] The compositions according to the present invention may be
in unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0340] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g. conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the present
invention. The tablets or pills of the novel composition can be
coated or otherwise compounded to provide a dosage form affording
the advantage of prolonged action. For example, the tablet or pill
can include an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two
components can be separated by an enteric layer that serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0341] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently include between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0342] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can include
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0.
[0343] The emulsion compositions can be those prepared by mixing a
EFNA4 antibody or a EFNA4 antibody-drug conjugate with
INTRALIPID.TM. or the components thereof (soybean oil, egg
phospholipids, glycerol and water).
[0344] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some aspects of the
invention, the compositions are administered by the oral or nasal
respiratory route for local or systemic effect. Compositions in
preferably sterile pharmaceutically acceptable solvents may be
nebulised by use of gases. Nebulised solutions may be breathed
directly from the nebulising device or the nebulising device may be
attached to a face mask, tent or intermittent positive pressure
breathing machine. Solution, suspension or powder compositions may
be administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0345] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
including the EFNA4 antibody or the EFNA4 antibody-drug conjugate
as described herein and instructions for use in accordance with any
of the methods of the invention described herein. Generally, these
instructions include a description of administration of the EFNA4
antibody or the EFNA4 antibody-drug conjugate for the above
described therapeutic treatments.
[0346] The instructions relating to the use of the EFNA4 antibodies
or the EFNA4 antibody conjugates as described herein generally
include information as to dosage, dosing schedule, and route of
administration for the intended treatment. The containers may be
unit doses, bulk packages (e.g., multi-dose packages) or sub-unit
doses. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0347] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an EFNA4 antibody or EFNA4
antibody-drug conjugate. The container may further include a second
pharmaceutically active agent.
[0348] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit includes a
container and a label or package insert(s) on or associated with
the container.
[0349] IV.E. Dose and Administration
[0350] For in vitro and in vivo applications, EFNA4 antibody-drug
conjugates are provided or administered in an effective dosage. In
a clinical context, an effective dosage of drug, compound, or
pharmaceutical composition is an amount sufficient to accomplish
prophylactic or therapeutic treatment either directly or
indirectly. An effective dosage can be administered in one or more
administrations. An effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved. For detection of EFNA-positive cells
using the disclosed EFNA4 antibody-drug conjugates, a detectable
amount of a composition of the invention is administered to a
subject, i.e., a dose of the conjugate such that the presence of
the conjugate may be determined in vitro or in vivo.
[0351] For example, when administered to a cancer-bearing subject,
an effective amount includes an amount sufficient to elicit
anti-cancer activity, including cancer cell cytolysis, inhibition
of cancer cell proliferation, induction of cancer cell apoptosis,
reduction of cancer cell antigens, delayed tumor growth, and/or
inhibition of metastasis. Tumor shrinkage is well accepted as a
clinical surrogate marker for efficacy. Another well accepted
marker for efficacy is progression-free survival. EFNA4
antibody-drug conjugates generally demonstrate at least a 25%
improvement in key efficacy parameters, such as improvement in
median survival, time to tumor progression, and overall response
rate.
[0352] The EFNA4 antibody or the EFNA4 antibody-drug conjugates can
be administered to an individual via any suitable route. It should
be understood by persons skilled in the art that the examples
described herein are not intended to be limiting but to be
illustrative of the techniques available. Accordingly, in some
aspects of the invention, the EFNA4 antibody or the EFNA4 antibody
conjugate is administered to an individual in accord with known
methods, such as intravenous administration, e.g., as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerebrospinal, intracranial, transdermal,
subcutaneous, intra-articular, sublingually, intrasynovial, via
insufflation, intrathecal, oral, inhalation or topical routes.
Administration can be systemic, e.g., intravenous administration,
or localized. Commercially available nebulizers for liquid
formulations, including jet nebulizers and ultrasonic nebulizers
are useful for administration. Liquid formulations can be directly
nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, the EFNA4 antibody or the EFNA4
antibody-drug conjugate can be aerosolized using a fluorocarbon
formulation and a metered dose inhaler, or inhaled as a lyophilized
and milled powder.
[0353] In some aspects of the invention, the EFNA4 antibody or the
EFNA4 antibody-drug conjugate is administered via site-specific or
targeted local delivery techniques. Examples of site-specific or
targeted local delivery techniques include various implantable
depot sources of the EFNA4 antibody or the EFNA4 antibody-drug
conjugate or local delivery catheters, such as infusion catheters,
indwelling catheters, or needle catheters, synthetic grafts,
adventitial wraps, shunts and stents or other implantable devices,
site specific carriers, direct injection, or direct application.
See, e.g. PCT International Publication No. WO 2000/53211 and U.S.
Pat. No. 5,981,568.
[0354] EFNA4 antibodies or the EFNA4 antibody-drug conjugates as
described herein can be administered using any suitable method,
including by injection (e.g., intraperitoneally, intravenously,
subcutaneously, intramuscularly, etc.). The EFNA4 antibody or the
EFNA4 antibody-drug conjugate can also be administered via
inhalation, as described herein. Generally, for administration of
an EFNA4 antibody and an EFNA4 antibody-drug conjugate, an initial
candidate dosage can be about 2 mg/kg. For the purpose of the
present invention, a typical daily dosage might range from about
any of 3 .mu.g/kg to 30 .mu.g/kg to 300 .mu.g/kg to 3 mg/kg, to 30
mg/kg, to 100 mg/kg or more, depending on the factors mentioned
above. For example, dosage of about 1 mg/kg, about 2.5 mg/kg, about
5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. For
repeated administrations over several days or longer, depending on
the disorder, the treatment is sustained until a desired
suppression of symptoms occurs or until sufficient therapeutic
levels are achieved, for example, to inhibit or delay tumor
growth/progression or metatstasis of cancer cells. An exemplary
dosing regimen includes administering an initial dose of about 2
mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of
the EFNA4 antibody or EFNA4 antibody-drug conjugate, or followed by
a maintenance dose of about 1 mg/kg every other week. Other
exemplary dosing regimen include administering increasing doses
(e.g., initial dose of 1 mg/kg and gradual increase to one or more
higher doses every week or longer time period). Other dosage
regimens may also be useful, depending on the pattern of
pharmacokinetic decay that the practitioner wishes to achieve. For
example, in some aspects of the invention, dosing from one to four
times a week is contemplated. In other aspects, dosing once a month
or once every other month or every three months is contemplated, as
well as weekly, bi-weekly and every three weeks. The progress of
this therapy may be easily monitored by conventional techniques and
assays. The dosing regimen (including the EFNA4 antibody or the
EFNA4 antibody-drug conjugate used) can vary over time.
[0355] For the purpose of the present invention, the appropriate
dosage of an EFNA4 antibody or an EFNA4 antibody-drug conjugate
will depend on the EFNA4 antibody or the EFNA4 antibody-drug
conjugate (or compositions thereof) employed, the type and severity
of symptoms to be treated, whether the agent is administered for
therapeutic purposes, previous therapy, the patient's clinical
history and response to the agent, the patient's clearance rate for
the administered agent, and the discretion of the attending
physician. The clinician may administer an EFNA4 antibody or an
EFNA4 antibody-drug conjugate until a dosage is reached that
achieves the desired result and beyond. Dose and/or frequency can
vary over course of treatment, but may stay constant as well.
Empirical considerations, such as the half-life, generally will
contribute to the determination of the dosage. For example,
antibodies that are compatible with the human immune system, such
as humanized antibodies or fully human antibodies, may be used to
prolong half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of symptoms, e.g.,
tumor growth inhibition or delay, etc. Alternatively, sustained
continuous release formulations of EFNA4 antibodies or EFNA4
antibody-drug conjugates may be appropriate. Various formulations
and devices for achieving sustained release are known in the
art.
[0356] In some aspects of the invention, dosages for an EFNA4
antibody or an EFNA4 antibody-drug conjugate may be determined
empirically in individuals who have been given one or more
administration(s) of the EFNA4 antibody or the EFNA4 antibody-drug
conjugate. Individuals are given incremental dosages of an EFNA4
antibody or an EFNA4 antibody-drug conjugate. To assess efficacy,
an indicator of the disorder can be followed.
[0357] Administration of an EFNA4 antibody or an EFNA4
antibody-drug conjugate in accordance with the method in the
present invention can be continuous or intermittent, depending, for
example, upon the recipient's physiological disorder, whether the
purpose of the administration is therapeutic or prophylactic, and
other factors known to skilled practitioners. The administration of
an EFNA4 antibody or an EFNA4 antibody-drug conjugate may be
essentially continuous over a preselected period of time or may be
in a series of spaced doses.
[0358] IV.F. Combination Therapies
[0359] In some aspects of the invention, the methods described
herein further include a step of treating a subject with an
additional form of therapy. In some aspects, the additional form of
therapy is an additional anti-cancer therapy including, but not
limited to, chemotherapy, radiation, surgery, hormone therapy,
and/or additional immunotherapy.
[0360] The disclosed EFNA4 antibody-drug conjugates may be
administered as an initial treatment, or for treatment of disorders
that are unresponsive to conventional therapies. In addition, the
EFNA4 antibody-drug conjugates may be used in combination with
other therapies (e.g., surgical excision, radiation, additional
anti-cancer drugs etc.) to thereby elicit additive or potentiated
therapeutic effects and/or reduce hepatocytotoxicity of some
anti-cancer agents. EFNA4 antibody-drug conjugates of the invention
may be co-administered or co-formulated with additional agents, or
formulated for consecutive administration with additional agents in
any order.
[0361] Representative agents useful for combination therapy include
any of the drugs described herein above as useful for preparation
of EFNA4 antibody-drug conjugates under the subheading "Drugs."
EFNA4 antibody-drug conjugates of the invention may also be used in
combination with other therapeutic antibodies and antibody-drug
conjugates, including anti-EFNA antibodies other than the disclosed
anti-EFNA antibodies, as well as antibodies and conjugates
targeting a different antigen. Representative antibodies, which may
be used alone or as an antibody-drug conjugate, include anti-5T4
antibodies (e.g., A1, A2, and A3), anti-CD19 antibodies, anti-CD20
antibodies (e.g., RITUXAN.RTM., ZEVALIN.RTM., BEXXAR.RTM.),
anti-CD22 antibodies, anti-CD33 antibodies (e.g., MYLOTARG.RTM.),
anti-CD33 antibody-drug conjugates, anti-Lewis Y antibodies (e.g.,
Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g.,
HERCEPTIN.RTM. (trastuzumab), MDX-210, OMNITARG.RTM. (pertuzumab,
rhuMAb 2C4)), anti-CD52 antibodies (e.g., CAMPATH.RTM.), anti-EGFR
antibodies (e.g., ERBITUX.RTM. (cetuximab), ABX-EGF (panitumumab)),
anti-VEGF antibodies (e.g., AVASTIN.RTM. (bevacizumab)),
anti-DNA/histone complex antibodies (e.g., ch-TNT-1/b), anti-CEA
antibodies (e.g., CEA-Cide, YMB-1003) hLM609, anti-CD47 antibodies
(e.g., 6H9), anti-VEGFR2 (or kinase insert domain-containing
receptor, KDR) antibodies (e.g., IMC-1C11), anti-Ep-CAM antibodies
(e.g., ING-1), anti-FAP antibodies (e.g., sibrotuzumab), anti-DR4
antibodies (e.g., TRAIL-R), anti-progesterone receptor antibodies
(e.g., 2C5), anti-CA19.9 antibodies (e.g., GIVAREX.RTM.) and
anti-fibrin antibodies (e.g., MH-1).
[0362] The disclosed EFNA4 antibody-drug conjugates may also be
administered together with one or more combinations of cytotoxic
agents as part of a treatment regimen. Useful cytotoxic
preparations for this purpose include CHOPP (cyclophosphamide,
doxorubicin, vincristine, prednisone and procarbazine); CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone); COP
(cyclophosphamide, vincristine, prednisone); CAP-BOP
(cyclophosphamide, doxorubicin, procarbazine, bleomycin,
vincristine and prednisone); m-BACOD (methotrexate, bleomycin,
doxorubicin, cyclophosphamide, vincristine, dexamethasone, and
leucovorin; ProMACE-MOPP (prednisone, methotrexate, doxorubicin,
cyclophosphamide, etoposide, leukovorin, mechloethamine,
vincristine, prednisone and procarbazine); ProMACE-CytaBOM
(prednisone, methotrexate, doxorubicin, cyclophosphamide,
etoposide, leukovorin, cytarabine, bleomycin and vincristine);
MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,
prednisone, bleomycin and leukovorin); MOPP (mechloethamine,
vincristine, prednisone and procarbazine); ABVD
(adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine);
MOPP (mechloethamine, vincristine, prednisone and procarbazine)
alternating with ABV (adriamycin/doxorubicin, bleomycin,
vinblastine); MOPP (mechloethamine, vincristine, prednisone and
procarbazin) alternating with ABVD (adriamycin/doxorubicin,
bleomycin, vinblastine and dacarbazine); ChIVPP (chlorambucil,
vinblastine, procarbazine, prednisone); IMVP-16 (ifosfamide,
methotrexate, etoposide); MIME (methyl-gag, ifosfamide,
methotrexate, etoposide); DHAP (dexamethasone, high-dose cytaribine
and cisplatin); ESHAP (etoposide, methylpredisolone, HD cytarabine,
and cisplatin); CEPP(B) (cyclophosphamide, etoposide, procarbazine,
prednisone and bleomycin); CAMP (lomustine, mitoxantrone,
cytarabine and prednisone); and CVP-1 (cyclophosphamide,
vincristine and prednisone); DHAP (cisplatin, high-dose cytarabine
and dexamethasone); CAP (cyclophosphamide, doxorubicin, cisplatin);
PV (cisplatin, vinblastine or vindesine); CE (carboplatin,
etoposide); EP (etoposide, cisplatin); MVP (mitomycin, vinblastine
or vindesine, cisplatin); PFL (cisplatin, 5-fluorouracil,
leucovorin); IM (ifosfamide, mitomycin); IE (ifosfamide,
etoposide); IP (ifosfamide, cisplatin); MIP (mitomycin, ifosfamide,
cisplatin); ICE (ifosfamide, carboplatin, etoposide); PIE
(cisplatin, ifosfamide, etoposide); Viorelbine and cisplatin;
Carboplatin and paclitaxel; CAV (cyclophosphamide, doxorubicin,
vincristine); CAE (cyclophosphamide, doxorubicin, etoposide); CAVE
(cyclophosphamide, doxorubicin, vincristine, etoposide); EP
(etoposide, cisplatin); and CMCcV (cyclophosphamide, methotrexate,
lomustine, vincristine).
[0363] EFNA4 antibody-drug conjugates may be used in combination
with systemic anti-cancer drugs, such as epithilones (BMS-247550,
Epo-906), reformulations of taxanes (Abraxane, Xyotax),
microtubulin inhibitors (MST-997, TTI-237), or with targeted
cytotoxins such as CMD-193 and SGN-15. Additional useful
anti-cancer agents include TAXOTERE.RTM., TARCEVA.RTM., GEMZAR.RTM.
(gemcitabine), 5-FU, AVASTIN.RTM. ERBITUX.RTM., TROVAX.RTM.,
anatumomab mafenatox, letrazole, docetaxel, and anthracyclines.
[0364] For combination therapies, an EFNA4 antibody-drug conjugate
and/or one or more additional therapeutic or diagnostic agents are
administered within any time frame suitable for performance of the
intended therapy or diagnosis. Thus, the single agents may be
administered substantially simultaneously (i.e., as a single
formulation or within minutes or hours) or consecutively in any
order. For example, single agent treatments may be administered
within about 1 year of each other, such as within about 10, 8, 6,
4, or 2 months, or within 4, 3, 2 or 1 week(s), or within about 5,
4, 3, 2 or 1 day(s). The administration of an EFNA4 antibody-drug
conjugate in combination with a second therapeutic agent preferably
elicits a greater effect than administration of either alone.
[0365] In some aspects of the invention, the additional form of
therapy includes administering one or more therapeutic agent in
addition to the EFNA4 antibodies or the EFNA4 antibody-drug
conjugates as described herein. The therapeutic agents include, but
are not limited to, a second antibody (e.g., an anti-VEGF antibody,
an anti-HER2 antibody, anti-CD25 antibody, and/or an anti-CD20
antibody), an angiogenesis inhibitor, a cytotoxic agent, an
anti-inflammatory agent (e.g., paclitaxel, docetaxel, cisplatin,
doxorubicin, prednisone, mitomycin, progesterone, tamoxifen, or
fluorouracil).
[0366] In some aspects of the invention, more than one EFNA4
antibody or EFNA4 antibody-drug conjugate may be present. At least
one, at least two, at least three, at least four, at least five
different or more EFNA4 antibody or EFNA4 antibody-drug conjugate
can be present. Generally, those EFNA4 antibodies or EFNA4
antibody-drug conjugates may have complementary activities that do
not adversely affect each other. For example, one or more of the
following EFNA4 antibody may be used: a first EFNA4 antibody
directed to one epitope on EFNA4 and a second EFNA4 antibody
directed to a different epitope on EFNA4.
[0367] The disclosed combination therapies may elicit a synergistic
therapeutic effect, i.e., an effect greater than the sum of their
individual effects or therapeutic outcomes. Measurable therapeutic
outcomes are described herein. For example, a synergistic
therapeutic effect may be an effect of at least about two-fold
greater than the therapeutic effect elicited by a single agent, or
the sum of the therapeutic effects elicited by the single agents of
a given combination, or at least about five-fold greater, or at
least about ten-fold greater, or at least about twenty-fold
greater, or at least about fifty-fold greater, or at least about
one hundred-fold greater. A synergistic therapeutic effect may also
be observed as an increase in therapeutic effect of at least 10%
compared to the therapeutic effect elicited by a single agent, or
the sum of the therapeutic effects elicited by the single agents of
a given combination, or at least 20%, or at least 30%, or at least
40%, or at least 50%, or at least 60%, or at least 70%, or at least
80%, or at least 90%, or at least 100%, or more. A synergistic
effect is also an effect that permits reduced dosing of therapeutic
agents when they are used in combination.
[0368] As used throughout the detailed description, the term
"about" means a value +/-1% of the value following the term
"about," unless otherwise indicated.
EXAMPLES
[0369] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Example 1
Generation of Anti-EFNA4 Antibodies
[0370] As described in International Publication No. WO2012/118547,
EFNA4 murine antibodies were produced by inoculation with
hEFNA4-ECD-Fc, hEFNA4-ECD-His, whole cell BALB/c 3T3 cells over
expressing EFNA4 or the plasma preps prepared as set forth herein
(ECD--extracellular domain). Immunogens were all prepared using
commercially available starting materials (e.g., Recombinant Human
ephrin-A4 Fc Chimera, CF R&D systems #369-EA-200) and/or
techniques well known to those skilled in the art.
[0371] EFNA4 murine antibodies were generated by immunizing female
mice (BaIb/c, CD-I, FVB) with various preparations of EFNA4
antigen. Immunogens included Fc constructs or His tagged human
EFNA4, membrane fractions extracted from 10.sup.7 over expressing
EFNA4 293 cells or whole 3T3 cells over expressing human EFNA4 on
the surface. Mice were immunized via footpad route for all
injections. 10 .mu.g of EFNA4 immunogen or 1.times.10.sup.6 cells
or cell equivalents emulsified with an equal volume of TITERMAX or
alum adjuvant were used for immunization. After immunization mice
were euthanized, and draining lymph nodes (popliteal and inguinal,
if enlarged) were dissected out and used as a source for antibody
producing cells. Lymphocytes were released by mechanical disruption
of the lymph nodes using a tissue grinder.
[0372] One of two fusion protocols was used. In the first
electrofusion with a Genetronic device was performed followed by
plating and screening of the polyclonal hybridomas with a
subsequent subcloning to generate monoclonal hybridomas. In the
second ectrofusion with a BTX instrument was performed followed by
growth of the hybridoma library in bulk and single cell deposition
of the hybridomas with a subsequent screen of the clones.
[0373] Genetronic device fusion protocol: The fusion was performed
by mixing a single cell suspension of B cells with non-secreting
P3.times.63Ag8.653 myeloma cells purchased from (ATCC CRL-1580;
Kearney et al, J. Immunol. 123: 1548-1550 (1979)) at a ratio of
1:1. The cell mixture was gently pelleted by centrifugation at 800
g. After complete removal of the supernatant, the cells were
treated with 2-4 mL of Pronase solution for no more than 2 minutes.
Electrofusion was performed using a fusion generator, model ECM2001
(Genetronic, Inc.). Cells were plated at 2.times.10.sup.4/well in
flat bottom microtiter plates, followed by two weeks incubation in
selective HAT medium (Sigma, CRL P-7185). Individual wells were
then screened by ELISA and FACS for anti-human EFNA4 monoclonal IgG
antibodies.
[0374] ELISA microtiter plates were coated with purified
recombinant EFNA4 His fusion proteins from transfected 293 cells at
100 ng/well in carbonate buffer. Plates incubated at 4.degree. C.
overnight than blocked with 200 .mu.//well of 3% BSA in PBS/Tween
(0.05%). Supernatant from hybridoma plates were added to each well
and incubated for 1-2 hours at ambient temperature. The plates were
washed with PBS/Tween and then incubated with Goat anti mouse IgG,
Fc Fragment Specific conjugated with horseradish proxidase (HRP)
Jackson ImmunoResearch) for one hour at room temperature. After
washing, the plates were developed with TMB substrate (Thermo
Scientific 34028) and analyzed by spectrophotometer at OD 450.
[0375] EFNA4 secreted hybridoma from positive wells were,
rescreened and subcloned by limited dilution or single cell FACS
sorting. Sub cloning was performed on selected antigen-positive
wells using limited dilution plating. Plates were visually
inspected for the presence of single colony growth and supernatants
from single colony wells then screened by antigen-specific ELISAs
described above and FACS confirmation as described below. The
resulting clonal populations were expanded and cryopreserved in
freezing medium (90% FBS, 10% DMSO) and stored in liquid nitrogen.
This fusion from mice immunized with EFNA4 yielded murine
monoclonal antibodies reactive for EFNA4 using the ELISA protocol
described above.
[0376] BTX instrument fusion protocol: A single cell suspension of
B cells were fused with non-secreting P3.times.63Ag8.653 myeloma
cells at a ratio of 1:1 by electrofusion. Electrofusion was
performed using the Hybrimune System, model 47-0300, (BTX Harvard
Apparatus). Fused cells were resuspended in hybridoma selection
medium supplemented with Azaserine (Sigma #A9666) (DMEM (Cellgro
cat#15-017-CM) medium containing, 15% Fetal Clone I serum
(Hyclone), 10% BM Condimed (Roche Applied Sciences), 1 mM sodium
pyruvate, 4 mM L-glutamine, 100 IU Penicillin-Streptomycin, 50
.mu.M 2-mercaptoefhanol, and 100 .mu.M hypoxanthine) and then
plated in four T225 flasks at 90 ml selection medium per flask. The
flasks are then placed in a humidified 37.degree. C. incubator
containing 5% CO.sub.2 and 95% air for 6-7 days.
[0377] At 6-7 days of growth the library is plated at 1 cell per
well in 48 Falcon 96 well U-bottom plates using the Aria I cell
sorter. Briefly culture medium containing 15% Fetal Clone I serum
(Hyclone), 10% BM-Condimed (Roche Applied Sciences), 1 mM sodium
pyruvate, 4 mM L-glutamine, 100 IU Penecillin-Streptamycin, 50
.mu.M 2-mercaptoethanol, and 100 .mu.M hypoxanthine is plated at
200 ul per well in 48 Falcon 96 well U-bottom plates. Viable
hybridomas are placed at 1 cell per well using the Aria I cell
sorter and cultured for 10-11 days and the supernatants are assayed
for antibodies reactive by FACS or ELISA for EFNA4.
[0378] Growth positive hybridomas wells secreting mouse
immunoglobulins were screened for murine EFNA4 specificity using an
ELISA assay similar to that described above. Briefly, 96 well
plates (VWR, 610744) were coated with 1 .mu.g/mL murine EFNA4-His
in sodium carbonate buffer overnight at 4.degree. C. The plates
were washed and blocked with 2% FCS-PBS for one hour at 37.degree.
C. and used immediately or kept at 4.degree. C. Undiluted hybridoma
supernatants were incubated on the plates for one hour at RT. The
plates are washed and probed with HRP labeled goat anti-mouse IgG
diluted 1:10,000 in 1% BSA-PBS for one hour at RT. The plates are
then incubated with substrate solution as described above and read
at OD 450. The amino acid sequences and associated nucleic acid
sequences (CDRs underlined) of exemplary murine antibodies E2, E5,
E8, E15, E22, E31, E47, E60, E73, E76, E91 and E105 that bound
human EFNA4 with high affinity are provided in International
Publication No. WO2012/118547.
[0379] Binding characteristics of various murine anti-EFNA4
antibodies of the present invention are shown in Table 3. The
antibodies exhibited relatively high affinities in the nanomolar
range and bind to at least three different bins or epitopes on the
EFNA4 protein. Most anti-EFNA4 antibodies reacted only with antigen
where disulfide bonds are intact (NR), while E22 and E91 reacted
with both non-reduced and reduced antigen (NR/R). E22 and E91
recognized the sequence QRFTPFSLGFE (SEQ ID NO: 137 and RLLRGDAVVE
(SEQ ID NO: 138), respectively. Further, E5, E15, E91 and E105 were
cross-reactive with mouse EFNA4 and all antibodies cross-reacted
with highly similar cynomolgus EFNA4. The ability of the antibodies
to neutralize (i.e. block receptor ligand interaction, specifically
EFNA4-EphA2) and/or internalize, along with the ability to kill
cells is also provided.
TABLE-US-00003 TABLE 3 Affinity Western Mouse Cyno Clone Bin (nM)
Reactivity XR XR Neutralizing Internalizing Killing E2 A 20.sup.F
NR No ND No Yes Yes E5 B 0.3.sup.B NR Yes Yes No Yes Yes E15 B
4.8.sup.B NR Yes Yes ND Yes Yes E22 A 3.1.sup.B NR/R No Yes No Yes
Yes E31 A 11.sup.B NR ND ND Yes Yes Yes E47 C <0.1.sup.B NR No
Yes Yes Yes Yes E76 A 0.4.sup.F NR ND ND No Yes ND E91 B 0.2.sup.B
NR/R Yes Yes No Yes Yes E105 B 16.sup.F ND Yes Yes ND Yes Yes
.sup.BBiacore affinity; .sup.FForteBIO in-house comparison; ND: not
determined
Example 2
Humanization of Anti-EFNA4 Antibodies
[0380] As further described in International Publication No.
WO2012/118547, four of the murine antibodies from Example 1 were
humanized using complementarity determining region (CDR) grafting.
Human frameworks for heavy and light chains were selected based on
sequence and structure similarity with respect to functional human
germline genes. Structural similarity was evaluated by comparing
the mouse canonical CDR structure to human candidates with the same
canonical structures as described in Chothia et al. (supra).
[0381] More particularly murine antibodies E5, E15, E22 and E47
were humanized using a computer-aided CDR-grafting method (Abysis
Database, UCL Business Pic.) and standard molecular engineering
techniques to generate humanized antibodies E5, E15, E22 and E47,
hereinafter huE5, huE15, huE22 and huE47, respectively. The human
framework regions of the variable regions were selected based on
their highest sequence homology to the mouse framework sequence and
its canonical structure. For the purposes of the analysis the
assignment of amino acids to each of the CDR domains is in
accordance with the Kabat et al. numbering. Several humanized
antibody variants were made in order to generate the optimal
humanized antibody with the humanized antibodies generally
retaining the antigen-binding complementarity-determining regions
(CDRs) from the mouse hybridoma in association with human framework
regions. HuE15, huE22 and huE47 mAbs bound to EFNA4 antigen with
similar affinity to their murine counterpart and huE5 bound with
slightly lower affinity as measured using the Biacore system.
[0382] Molecular engineering procedures were conducted using
art-recognized techniques. Total mRNA was extracted from the
hybridomas according to the manufacturer's protocol (Trizol.RTM.
Plus RNA Purification System, Life Technologies). A primer mix
including thirty-two mouse specific 5' leader sequence primers,
designed to target the complete mouse repertoire, was used in
combination with 3' mouse C.gamma.1primer to amplify and sequence
the variable region of the antibody heavy chains. Similarly
thirty-two 5' Vk leader sequence primer mix designed to amplify
each of the Vk mouse families combined with a single reverse primer
specific to the mouse kappa constant region were used to amplify
and sequence the kappa light chain. The V.sub.H and V.sub.L
transcripts were amplified from 100 ng total RNA using reverse
transcriptase polymerase chain reaction (RT-PCR).
[0383] A total of eight RT-PCR reactions were run for each
hybridoma: four for the V kappa light chain and four for the V
gamma heavy chain (.gamma.1). The QIAGEN One Step RT-PCR kit was
used for amplification, (Qiagen, Inc.). The extracted PCR products
were directly sequenced using specific V region primers. Nucleotide
sequences were analyzed using IMGT to identify germline V, D and J
gene members with the highest sequence homology. The derived
sequences were compared to known germline DNA sequences of the Ig
V- and J-regions derived from an analysis of the VBASE2 database
(Retter et al., supra) and by alignment of V.sub.H and VL genes to
the mouse germ line database.
[0384] From the nucleotide sequence information, data regarding V,
D and J gene segments of the heavy and light chains of E5, E15, E22
and E47 were obtained. Based on the sequence data new primer sets
specific to the leader sequence of the Ig V.sub.H and V.sub.K chain
of the antibodies were designed for cloning of the recombinant
monoclonal antibody. Subsequently the V-(D)-J sequences were
aligned with mouse Ig germ line sequences.
[0385] Heavy chain and light chain genes of E5, E15, E22 and E47
were identified and the results are summarized in Table 4
below.
TABLE-US-00004 TABLE 4 Mouse Clone Isotype VH DH JH VL JL E5 IgG1/K
IGHV2-6 None JH3 IGKV6-15 JK2 E15 IgG1/K IGHV5-6 DSP2.9 JH3 IGKV6-b
JK5 E22 IgG2b/K VhJ558 DFL16.1e JH4 IGKV1-110 JK1 E47 IgG1/K
IGHV1-26 P1inv JH2 IGKV21-7 JK1
[0386] The obtained heavy and light chain sequences from all four
clones were aligned to the functional human variable region
sequences and reviewed for homology and canonical structure. The
result the heavy and light chain analysis are shown below in Tables
5 and 6, respectively.
TABLE-US-00005 TABLE 5 % Homology Human Human Human to human germ %
Homology to Clone VH DH JH line sequence mouse sequence E5 VH3-66
IGHD2 JH4 82 75 E15 VH3-21 IGHD5-5 JH4 88 88 E22 VH1-18 IGHD5-24
JH6 87 83 E47 VH1-46 IGHD3-10 JH4 91 76
TABLE-US-00006 TABLE 6 % Homology Human % Homology to human to
mouse Clone Human VK JK germ line sequence sequence E5 L1 JK2 86 79
E15 A27 JK4 89 76 E22 A18b JK1 89 91 E47 L6 JK4 87 84
[0387] The germ line selection and CDR grafting processes appeared
to provide antibodies that generally retained their binding
characteristics, there was little need to insert murine residues in
most of the constructs. However, in huE15 the heavy chain residue
68 was back mutated from Thr (T) to Lys (K) to improve the antibody
characteristics.
[0388] The amino acid sequences and associated nucleic acid
sequence of huE5, huE15, huE22 and huE47 are shown above in Table 2
above. The amino acid sequences of the VH region for huE5, huE15,
huE22 and huE47 are shown in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO:
13 and SEQ ID NO: 39 respectively, with the corresponding nucleic
acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO:
14 and SEQ ID NO: 40. The amino acid sequence of the kappa VL
region of huE5, huE15, huE22 and huE47 are shown in SEQ ID NO: 7,
SEQ ID NO: 11, SEQ ID NO: 27 and SEQ ID NO: 53 respectively, with
the corresponding nucleic acid sequences set forth in SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 28 and SEQ ID NO: 54.
Example 3
Expression of Humanized Anti-EFNA4 Antibodies
[0389] The anti-EFNA4 antibodies huE5, huE15, huE22 and huE47 were
expressed and isolated using art recognized techniques and
described in International Publication No. WO2012/118547. To that
end synthetic humanized variable DNA fragments (Integrated DNA
Technologies) of both heavy chains were cloned into human IgGI
expression vector. The variable light chain fragments were cloned
into human C-kappa expression vector. Antibodies were expressed by
co-transfection of the heavy and the light chain into CHO
cells.
[0390] More particularly, for antibody production, directional
cloning of the murine and humanized variable gene PCR products into
human immunoglobulin expression vectors was undertaken. All primers
used in Ig gene-specific PCRs included restriction sites (AgeI and
XhoI for IgH, XmaI and DraIII for IgK, which allowed direct cloning
into expression vectors containing the human IgGI, and IGK constant
regions, respectively. In brief, PCR products were purified with
Qiaquick PCR purification kit (Qiagen, Inc.) followed by digestion
with AgeI and XhoI (IgH), XmaI and DraIII (IgK), respectively.
Digested PCR products were purified prior to ligation into
expression vectors. Ligation reactions were performed in a total
volume of 10 .mu.L with 200U T4-DNA Ligase (New England Biolabs),
7.5 .mu.L of digested and purified gene-specific PCR product and 25
ng linearized vector DNA. Competent E. coli DH1OB bacteria (Life
Technologies) were transformed via heat shock at 42.degree. C. with
3 .mu.L ligation product and plated onto ampicillin plates (100
.mu.g/mL). The AgeI-EcoRI fragment of the V.sub.H region was than
inserted into the same sites of pEE6.4HulgGI expression vector
while the synthetic XmaI-DraIII VK insert was cloned into the
XmaI-DraIII sites of -the respective pEE12.4Hu-Kappa expression
vector.
[0391] Cells producing humanized antibodies were generated by
transfection of HEK 293 cells with the appropriate plasmids using
293fectin. Plasmid DNA was purified with QIAprep Spin columns
(Qiagen). Human embryonic kidney (HEK) 293T (ATCC No CRL-1 1268)
cells were cultured in 150mm plates (Falcon, Becton Dickinson)
under standard conditions in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% heat inactivated FCS, 100 .mu.g/mL
streptomycin, 100 .mu.g/mL penicillin G (all from Life
Technologies).
[0392] For transient transfections, cells were grown to 80%
confluency. Equal amounts of IgH and corresponding IgL chain vector
DNA (12.5 .mu.g of each vector DNA) was added to 1.5 mL Opti-MEM
mixed with 50 .mu.L HEK 293 transfection reagent in 1.5 mL
opti-MEM. The mix was incubated for 30 min at room temperature and
distributed evenly to the culture plate. Supernatants were
harvested three days after transfection, replaced by 20 mL of fresh
DMEM supplemented with 10% FBS and harvested again at day 6 after
transfection. Culture supernatants were cleared from cell debris by
centrifugation at 800.times.g for 10 min and stored at 4.degree. C.
Recombinant chimeric and humanized antibodies were purified with
Protein G beads (GE Healthcare).
Example 4
Binding Properties of Anti-EFNA4 Antibodies
[0393] As described in International Publication No. WO2012/118547,
binding characteristics of E15, E22 and E47 antibodies of the
present invention are shown in Table 7.
TABLE-US-00007 TABLE 7 mAb Mouse Ag Cyno Ag Hu Ag Affinity Hu Ag
Affinity Clone Bin Isotype Affinity Binding (Murine mAb) (Humanized
mAb) E15 B chimHuIgG1 3.4 nM + 2.7 nM 4.8 nM E22 A msIgG2b >100
nM + 3.1 nM 3.8 nM E47 C msIgG1 >100 nM + <0.1 nM <0.1
nM
[0394] Further properties were determined using ELISA to analyze
the binding specificity of anti-EFNA4 antibodies huE22 and huE47 to
Ephrin-A4 (EFNA4) along with homologous family members Ephrin-A1
(EFNA1) and Ephrin-A3 (EFNA3). Antibody binding was determined by
direct ELISA with Ephrin-A1-His, Ephrin-A3-huFc, Ephrin-A4-huFc and
Ephrin-A5-huFc (all from R&D) coated on BioOne Microlon
(Greiner) plates in PBS overnight at 4.degree. C. Biotin-labeled
huE22 and huE47 were incubated on the plate for 2.5 hours at room
temperature and Streptavidin HRP was used to develop signal from
biotin labeled antibody in the absence of interfering human IgG
signal. The data shown in FIG. 3A demonstrates that huE22 binds to
EFNA4 but not to EFNA1, EFNA3 or EFNA5. The data shown in FIG. 3B
demonstrates that huE47 binds to EFNA4 but not EFNA1 or EFNA3.
[0395] Binding of humanized anti-EFNA4 antibodies huE22 and huE47
was assessed on EFNA4 expressing cells by flow cytometry. Adherent
cells were dissociated using TrpLE Express (GIBCO cat #12604-021),
neutralized with cell culture media and counted. Cells were plated
into a U-bottom 96-well plate (BD Falcon cat #353077) with
5.times.10e5 cells/100 .mu.L media/well. The plate was centrifuged
at 300.times.g, 5 minutes, 4.degree. C. to pellet cells and the
supernatant was discarded. All reagents were kept on ice for the
following steps. Each pellet was resuspended in 100 .mu.L primary
humanized anti-EFNA4 antibody (huE22 or huE47) or negative control
antibody at 10 .mu.g/mL in 3% BSA in PBS. The plate was incubated
on ice for 30 minutes. The plate was centrifuged and the cell
pellets were washed in 200 .mu.L 3% BSA in PBS. Each cell pellet
was resuspended in 100 .mu.L of R-phycoerythrin (PE)-conjugated
goat anti-human IgG Fc fragment (Jackson ImmunoResearch cat
#109-115-098) that had been diluted 1:50 in 3% BSA in PBS. The
plate was incubated on ice for 30 minutes. The plate was
centrifuged and the cell pellets were washed in 200 .mu.L 3% BSA in
PBS. Each pellet was resuspended in 100 .mu.L 3% BSA in PBS and
transferred to a 5 mL polycarbonate tube (BD Falcon cat #352054)
containing 250 .mu.L 3% BSA in PBS. The samples were analyzed by
flow cytometry using 5 .mu.L 7AAD (B-D Pharmingen cat #51-68981 E)
per sample as a viability stain.
[0396] Antibody binding was measured in the manner described above
for HEK293T parental cells, which express very low levels of EFNA4,
and HEK293T-EFNA4 cells which are stable transfectants of HEK293T
with a vector that enables high expression of human EFNA4. Table 8
shows mean channel fluorescence (MCF) of antibodies binding to
EFNA4 expressing cell lines. The huE22 and huE47 antibodies
demonstrate specificity for the EFNA4 target by strongly binding to
the HEK293T-EFNA4 cells overexpressing cells but not to parental
HEK293T cells.
TABLE-US-00008 TABLE 8 Mean Channel Fluorescence (MCF) 10 .mu.g/ml
antibody Non- Expression binding Cell Line Level huE22 huE47
control IgG HEK293T Low 7.2 No data 6.9 parent expression of EFNA4
HEK293T- High 1521 576 3.3 EFNA4 expression of EFNA4
[0397] Endogenous EFNA4 expressing cancer cell lines were analyzed:
Breast cell lines BT-483, HCC202 and MX-1, Chronic Lymphocytic
Leukemia (CLL) cell line MEC1 and mantle cell lymphoma (MCL) cell
line Z138. The MCF values shown in Table 9 demonstrate the ability
of anti-EFNA4 antibodies, huE22 and huE47, to bind various cancer
cell lines with endogenous expression of the antigen.
TABLE-US-00009 TABLE 9 Mean Channel Fluorescent (MCF) 10 .mu.g/ml
antibody Non- binding Cell Line Tumor type huE22 huE47 control IgG
BT-483 Breast 17.7 24 3.1 HCC202 Breast 13.2 21.9 15.6 MX-1 Breast
12.8 21.1 3.6 MEC1 B-Chronic 24 33 7.2 Lymphocytic Leukemia (B-CLL)
Z138 Mantle Cell 44 44 7.4 Lymphoma (MCL)
Example 5
Internalization and In Vitro Cytotoxicity of Anti-EFNA4
Antibodies
[0398] The internalization of huE22 was assessed in a functional
assay in which cells were incubated with the primary antibody and a
secondary antibody (antigen-binding fragment, Fab) that is
conjugated to the saporin toxin. Cells were seeded in 96-well
plates, incubated overnight, and then exposed for four days to a
dose response of huE22 or human IgG1 control antibody each with
saporin-conjugated anti-human Fab fragment (Advanced Targeting
Systems catalog number IT-51) at a molar ratio of 1.5:1 of
secondary:primary antibodies. Cell viability was then measured with
the MTS assay (Promega catalog number G5430) and the primary
antibody concentration that inhibited cell viability by 50%
(IC.sub.50) relative to untreated cells was calculated by logistic
non-linear regression on GraphPad Prism software.
[0399] Cytotoxicity is achieved when the primary antibody such as
huE22 internalizes into the target cell and carries the
saporin-conjugated Fab fragment into the cell. As shown in Table
10, huE22 conferred saporin-mediated cytotoxicity to HEK293T-EFNA4
cells with .about.60-fold specificity over HEK293T parental cells,
while the control primary antibody did not confer significant
cytotoxicity to either cell line. Low levels of EFNA4 expressed in
HEK293T-parental cells may have led to huE22 generating a lower
IC.sub.50 value than the control antibody. These results
demonstrate that huE22 antibody internalized into the cells in a
target-dependent manner.
TABLE-US-00010 TABLE 10 Saporin-mediated cytotoxicity IC.sub.50
Values (ng/mL) Cell Line huE22 Control IgG HEK293T parent 174 375
HEK293T-EFNA4 3 360
Example 6
Conjugation and Purification Anti-EFNA4-AcBut-CM Antibody-Drug
Conjugates
A. Conjugation
[0400] In the present invention, anti-EFNA4 antibodies huE22 and
huE47 were conjugated to AcBut-N-acetyl-.gamma.-calicheamicin
dimethyl hydrazide (AcBut-CM) OSu ester, as shown in FIG. 4A, to
generate huE22-AcBut-CM ADC and huE47-AcBut-CM ADC as shown in FIG.
4B, wherein X can be any antibody, such as huE22 and huE47. The
AcBut-CM was conjugated to the anti-EFNA4 antibodies via lysine
residues to produce anti-EFNA4-AcBut-CM ADCs having a narrow
distribution of drug-to-antibody ratio (DAR), where about 70% to
80% of ADCs have a DAR between 3 and 5, and average DAR in the
range of about 3 to 5.
[0401] The conjugation reaction mixture for the generation of
huE22-AcBut-CM included 10 mg/ml or less of purified huE22 antibody
and AcBut-CM OSu ester at a molar ratio of 4-4.5 to 1. High
agitation was conducted during the addition of AcBut-CM to a mixing
vortex. The reaction pH was 8.3 and the concentrations of other
reaction components were as follows: 180 mM HEPES buffer, 41 mM
sodium decanoate, and 8% (v/v) ethanol. The reaction was conducted
at 33.degree. C. for 5 minutes. After the conjugation reaction was
completed, the reaction mixture was diluted slowly with 1.3 volumes
of 1M K.sub.2HPO.sub.4 adjusted to pH 8.5 with mixing. A similar
reaction mixture was used for the generation of huE47-AcBut-CM
ADC.
B. Purification
[0402] To purify, the above diluted reaction mixture was loaded in
two batches on a Butyl Sepharose-4 Fast Flow HIC column (GE
Healthcare) that was previously equilibrated in five column volumes
(cv) of 0.52M potassium phosphate buffer, pH 8.5, as shown in FIG.
5 (top line 280 nm and bottom line 310 nm). The protein loaded on
the column was 3.5 mg/ml of bed volume. The flow rate was 15
ml/minute through the sample loading and 22 ml/minute throughout
the wash and elution phase of the chromatography. This improved
gradient removes higher DAR ADCs that were bound to the column.
[0403] The unbound fraction during the loading was predominantly
reaction reagents and most of the unconjugated antibody, which was
discarded. The column was then washed with 0.3 cv of 0.52M
potassium phosphate buffer, pH 8.5, to remove any remaining
reagents. A step gradient with 1 cv from 0.52M to 0.4M potassium
phosphate buffer, pH 8.5 was then used to elute any loosely bound
unconjugated antibody along with low loaded anti-EFNA4-AcBut-CM, if
present. The main fraction was then eluted using a step gradient of
1 cv from 0.4M to 5 mM potassium phosphate buffer, pH 8.5, to
provide huE22-AcBut-CM having a DAR from 3 to 5, toward the end of
the gradient. If huE22-AcBut-CM conjugates with a higher DAR were
present, the fraction was eluted using a gradient of 2 cv of 5mM
potassium phosphate buffer, pH 8.5, and then an elution of pure
deionized water. Any huE22-AcBut-CM conjugates with a higher DAR
that remained bound after the deionized water elution were eluted
using 2 cv of 10 mM sodium hydroxide containing 20% ethanol.
[0404] Using capillary isoelectric focusing (cIEF; iCE280,
ProteinSimple) purified batches of huE22-ActBut-CM ADCs had a
narrow DAR distribution, where about 70% to 80%, preferably about
75% to 80%, of the ADCs had a DAR from 3 to 5. For example, FIG. 9
shows a purified batch of huE22-ActBut-CM ADCs had a narrow DAR
distribution, where about 78% of the ADCs had a DAR from 3 to 5.
The DAR is determined by UV spectroscopy, measuring absorbance at
280 nm and 310 nm.
[0405] Further, purified batches of huE22-ActBut-CM ADCs had an
average DAR in the range of about 3 to about 5. For example, a
purified batch of huE22-ActBut-CM ADCs had an average DAR of about
4, and more particularly the average DAR was about 4.6.
[0406] This improved conjugation and purification processes
generated ADCs having a DAR that was less than 6, and in some
aspects in the range of 3 to 5. Further, the processes generated a
narrower distribution of loading, for example, less heterogeneity
within the product. Improvements to the conjugation and
purification processes further included: 1) decreasing the AcBut-CM
to huE22 ratio to 4-4.5 to 1 to generate an ADC having a lower DAR,
2) conducting high agitation during addition of AcBut-CM to huE22
to generate ADCs with low amounts of unconjugated antibody (free
antibody), 3) reducing incubation time to 2-5 minutes, compared to
60-90 minutes, to provide low aggregates and 4) a reduction in
ethanol amount to 6-8% to provide low aggregates.
[0407] The purified pooled peaks from both batches were dialyzed
twice against a formulated buffer to facilitate storage in a frozen
state. The formulated buffer composition was 20 mM Tris, 7.5%
sucrose, 0.01% polysorbate 80, 10mM NaCl, pH 8.0. The purified
huE22-AcBut-CM ADC was characterized using the following: [0408]
Hydrophobic interaction chromatography (HIC; TSKgel Butyl-NPR,
Tosoh Bioscience, LLC) for the presence of unconjugated antibody,
as shown in FIG. 6, no free antibody was detected in the purified
huE22-AcBut-CM; [0409] Size exclusion chromatography (SEC; Acquity
UPLC BEH200 SEC, Waters) for presence of aggregates and dimer, as
shown in FIG. 7, there was 100% monomer, no aggregate or dimer was
detected; [0410] Reverse phase high-performance liquid
chromatography (HPLC-RP; Zorbax 300SB-CN, Agilent) for presence of
free drug, as shown in FIG. 8, no free drug was detected in the
purified huE22-AcBut-CM; and [0411] Capillary isoelectric focusing
(cIEF; iCE280, ProteinSimple) for the drug distribution profile, as
shown in FIG. 9, the desired narrow DAR distribution from 3 to 5
was about 78%, only traces of DAR 7 and higher species were present
and DAR 1 and unconjugated (DAR 0) species were not detected.
[0412] A similar purification process was used for the generation
of huE47-AcBut-CM ADC. FIG. 10 and Table 11 below show the effect
of freeze-thaw cycles on the stability of the purified
huE22-AcBut-CM antibody-drug conjugate. No significant changes were
observed on multiple freeze-thaws.
TABLE-US-00011 TABLE 11 huE22-AcBut-CM Antibody-Drug Conjugate %
Free Conditions Antibody % Monomer Free Drug T = 0 (not frozen) 0.2
100 n/a Freeze Thaw (-70.degree. C.) #1 0.1 100 None detected
Freeze Thaw (-70.degree. C.) #3 0.1 100 None detected Freeze Thaw
(-70.degree. C.) #6 0.1 100 None detected
[0413] FIG. 29 shows a comparison of analytical HICs for purified
ADC (2 mg) generated from a ratio of CM to huE22 Ab of 4 m/m and 6
m/m (top line 280 nm and bottom line 310 nm). The CM to huE22 Ab
ratio of 4 m/m generated ADCs having an average DAR of 4.3 (top
panel) and the CM to huE22 Ab ratio of 6 m/m generated ADCs having
an average DAR of 5.2 (bottom panel). The ADCs generated using 4
m/m had a higher yield of desired ADCs, characterized as low
hydrophobic monomers, and a lower yield of undesired ADCs,
characterized as highly hydrophobic aggregates. FIG. 30 shows a
further analytical HIC for purified ADCs generated from a ratio of
CM to huE22 Ab of 4 m/m (top line) and 6 m/m (bottom line). In
particular, the ADCs generated using 6 m/m (5.2 DAR preparation)
had an increased presence of highly hydrophobic species and
aggregation, which was lower in the 4 m/m (4.3 DAR preparation)
generated ADCs.
Example 7
In Vitro Binding and Cytotoxicity of Anti-EFNA4-AcBut-CM ADCs
A. Binding Assays
[0414] The binding constants of huE22 for human and cynomolgus
monkey EFNA4 proteins were determined by surface plasmon resonance
(SPR) using BIAcore 2000 (GE Healthcare) using recombinant EFNA4
extracellular domain that was fused to a histidine tag to
facilitate purification. The results indicated high affinity of
huE22 for both human and cynomolgus monkey EFNA4, see Table 12. The
cynomolgus monkey EFNA4 protein sequence is highly homologous to
human EFNA4 protein sequence, with 98% identity overall and 98%
identity in the extracellular domain. In contrast, no huE22 binding
to rat antigen was detected, and in separate studies no binding to
mouse antigen was detected. The lack of huE22 binding to rat or
mouse EFNA4 is likely explained by a non-conserved residue in the
rat and mouse EFNA4 sequences within the defined huE22 epitope.
[0415] The binding constants of huE22-AcBut-CM ADC for human and
cynomolgus monkey EFNA4 were determined in the same binding study
as huE22. As shown in Table 12, the ADC and unconjugated mAb
(huE22) had comparable binding constants to both human and
cynomolgus monkey antigen.
TABLE-US-00012 TABLE 12 Binding constants of huE22 mAb and
huE22-AcBut-CM ADC. kon koff Kd Antigen Species (M-1s-1) (s-1) (nM)
huE22 mAb Human 1.9E+06 3.3E-03 1.7 Cynomolgus monkey 3.8E+06
7.8E-03 2.1 Rat No binding detected at 400 nM antigen huE22-AcBut-
Human 2.6E+06 6.1E-03 2.3 CM ADC Cynomolgus monkey 4.2E+06 1.1E-02
2.7 Rat No binding detected at 400 nM antigen
[0416] Cell binding and internalization of huE22 and huE22-AcBut-CM
ADC were analyzed. Parental HEK293T cells (EFNA4 negative) and
HEK293T-EFNA4 (engineered to express high levels of human EFNA4)
were used. huE22 and huE22-AcBut-CM ADC demonstrated specific
binding to cells that expressed the EFNA4 antigen. Further, the
unconjugated huE22 and the huE22-AcBut-CM ADC exhibited comparable
binding to HEK293T-EFNA4 cells, and neither antibody bound to
parental HEK293T cells, see Table 13. Together with the SPR results
in Table 12 (above), these data indicate that the bioconjugation
process did not alter the binding characteristics of huE22.
TABLE-US-00013 TABLE 13 Cell binding of huE22 and huE22-AcBut-CM
ADC. Concentration 0.3 1.0 3.0 10.0 Cell Line Test Article .mu.g/mL
.mu.g/mL .mu.g/mL .mu.g/mL HEK293T huE22-AcBut- 6.8 6.1 8.2 13.4
parental CM ADC huE22 mAb 8.3 6.8 6.1 14.5 HEK293T- huE22-AcBut-
446 621 728 863 EFNA4 CM ADC huE22 mAb 446 606 739 856
B. Cytotoxicity Assay
[0417] Once the huE22-AcBut-CM ADC is internalized into the cell,
the release of calicheamicin elicits cytotoxicity. Internalizing
antibodies typically traffic to the lysosomal compartment for
degradation. A direct cytotoxicity assay was used to analyze
cytotoxic response of huE22-AcBut-CM. HEK293T-EFNA4 overexpressing
cells and the HEK293T parental (EFNA4 negative) cells were plated
into a clear flat-bottom tissue culture plate (BD Falcon cat #
353072) at 500 cells per 180 .mu.L of cell culture media per well.
The cells were incubated overnight at 37.degree. C. in a 5% CO2
incubator. On the following day the huE22-AcBut-CM and the negative
non-binding control hIgG1-AcBut-CM ADC were added to the cells as a
10 point concentration curve starting with 1 .mu.g/mL with half-log
dilutions in cell culture media in triplicate. The plate was
incubated in a 37.degree. C., 5% CO2 incubator for 96 hours. Cell
viability was then measured with the MTS assay (Promega CellTiter
96 Aqueous Non-Radioactive Cell Proliferation Assay cat #G5430)
according to the manufacturer's instructions. 40 .mu.L of the
combined MTS reagent was added to each well. The plate was
incubated in a 37.degree. C., 5% CO2 incubator for 1.5 hours. The
concentration that inhibited cell viability by 50% (IC.sub.50)
relative to untreated cells was calculated by logistic non-linear
regression on GraphPad Prism software, as show in Table 14. FIG. 21
shows HEK293T-EFNA4 cells exposed to various concentrations of
huE22-AcBut-CM (open diamonds) or hIgG-AcBut control ADC
(diagonal-hatched circles), and cell viability was measured and
normalized to that of the untreated cells.
[0418] As shown in Table 14 and FIG. 21, huE22-AcBut-CM elicited a
dose-dependent cytotoxic response in vitro against EFNA4
target-expressing cells and inhibited cell growth in a target- and
concentration dependent manner. When HEK293T-EFNA4 cells were
exposed to huE22-AcBut-CM, a concentration of 1 ng/mL ADC inhibited
cell growth by 50% (IC.sub.50), in contrast, the control ADC was
>150-fold less active. The data demonstrates the potent and
specific cytotoxic activity of huE22-AcBut-CM. The control ADC and
huE22-AcBut-CM ADC did not elicit cytotoxicity against HEK293T
parental cells that lack EFNA4 expression, which demonstrated
specificity and antigen dependence.
TABLE-US-00014 TABLE 14 Anti-EFNA4-AcBut-CM Cytotoxicity IC.sub.50
Values (ng/mL) Cell Line huE22-AcBut-CM Control-AcBut-CM HEK293T
parental 650 100 HEK293T-EFNA4 1 157
Example 8
In Vivo Efficacy of Anti-EFNA4 Antibody-Drug Conjugates
[0419] The effects of anti-EFNA4 huE22-AcBut-CM and huE47-AcBut-CM,
prepared according the conjugation and purification processes
described in Example 6, were further evaluated on the in vivo
growth of human tumor patient-derived xenografts (PDX). Primary
tumor resection samples were procured from clinical sites following
Institutional Review Board for the Protection of Human Subjects
approval and in accordance with HIPAA regulations.
[0420] The expression of the target EFNA4 in each model was
measured in two ways: reverse transcriptase polymerase chain
reaction (RT-PCR) using mRNA extracts and ELISA using protein
extracts. Table 15 shows the normalized mRNA and protein levels of
EFNA4 in the panel of tumor models. EFNA4 was detected in all
models tested, in other words both measurements exceeded those from
the tumor model MDA-MB-231.
TABLE-US-00015 TABLE 15 EFNA4 RT-PCR EFNA4 ELISA (normalized (ng
EFNA4/mg Tumor Type Tumor Model signal) protein) Breast cancer BR5
1.58 1.80 (TNBC) BR13 0.45 1.00 BR22 1.01 1.91 BR31 0.48 1.02
144580A1 3.86 1.74 MDA-MB-231 0.19 0.25 Ovarian cancer OV44 2.10
1.39 OV45 3.39 3.64 OV55 1.45 0.42 OV63 2.40 0.49 Lung LU86 0.85
0.45 LU80 ND ND Colorectal CR5 ND ND ND = Not Determined.
[0421] Tumor fragments were stored and shipped in Hypothermasol
(Biolife Solutions) on ice and were embedded in Matrigel (BD)
containing a proprietary mix of growth factors and implanted
subcutaneously into the mammary fatpad of female NOD/SCID mice
within 24 hours of resection. Mice were monitored for health status
daily and for tumor growth initially by visual inspection twice per
week. Once the tumors were palpable, measurements of tumor volume
began to track tumor growth and estimate cell doubling time. Tumor
volume was estimated using the equation V=(A*B.sup.2)/2 where A is
the long axis and B is the short axis. When tumor reached a volume
of 500 mm.sup.3 to 1,500 mm.sup.3, they were harvested for study
and for re-transplant. Depending on the line, mechanical and/or
chemical dissociation can be used to separate the individual cells
for passaging. Live cells were inoculated into naive animals with
10,000 to 50,000 cells per animals.
[0422] For efficacy studies, tumors were harvested from passaging
studies and cells were dissociated into single cell suspension.
Preparations were counted for live cells using Trypan blue
exclusion and 10,000 to 50,000 cells were inoculated per mouse in
Matrigel. To account for differential growth rates of PDX, at least
25% more animals were started to allow for minimal tumor volume
variance at randomization. Tumor growth was initially followed by
palpability with measurements beginning once tumor volumes reached
about 30 mm.sup.3. Studies were randomized based on tumor size once
a cohort of tumor-bearing mice reached 140-180 mm.sup.3; groups
ranged from 6-10 mice. Animals were dosed by intraperitoneal
injection twice a week for two weeks (Q4dx4) with ADC at various
dose levels, once a week for two weeks (Q7dx2) with doxorubicin at
1.5 mg/kg (maximum tolerated dose) or once a week for two weeks
with cisplatin at 5 mg/kg. Study groups were followed until
individual mice or entire group tumor measurements reached
1200mm.sup.3 when sacrifice was indicated in accordance with IACUC
protocol. For selected dosing studies, pharmacokinetic
submandibular bleeds were performed at 2 or 6 hours, 36 hours and
72 hours. A volume of 10 .mu.L of blood was immediately pipetted
into 90 .mu.L of HBS-P (GE Healthcare). Samples were stored at -80
C. prior to analysis. For each tumor measurement the tumor
volume.+-.standard error of the mean (SEM) is provided. GT=Group
Terminated due to large tumor size. ND=Not enough animals remaining
to make significant measurement with SEM. All studies included a
PBS vehicle and a control antibody-drug conjugate having a
non-binding hIgG1 antibody conjugated to the same linker-payload
being analyzed and with comparable drug-to-antibody ratio (DAR) and
DAR distribution.
A. Breast Cancer
[0423] Tables 16-20 and FIGS. 11-14 provide data which demonstrate
the efficacy of huE22-AcBut-CM and huE47-AcBut-CM in various
triple-negative breast cancer (TNBC) PDX tumor models. The results
obtained were unexpected because huE22-ActBut-CM demonstrated
superior efficacy in all TNBC PDX models compared to the treatment
with doxorubicin standard-of-care (SOC) which has a similar
mechanism of action to calicheamicin (DNA damaging agent).
[0424] Table 16 and FIG. 11 show the efficacy of huE22-AcBut-CM in
the Breast-5 (BR5) triple-negative breast cancer (TNBC) PDX.
Sustained regressions for more than 80 days were achieved with 0.27
mg/kg huE22-AcBut-CM, and dose levels as low as 0.036 mg/kg
elicited anti-tumor activity. In contrast, neither the control ADC
nor doxorubicin standard-of-care (SOC) impacted tumor growth.
TABLE-US-00016 TABLE 16 Efficacy of huE22-AcBut-CM in BR5 TNBC PDX
0.1 mg/kg 0.036 mg/kg 0.09 mg/kg 0.27 mg/kg Control- huE22- huE22-
huE22- Day Vehicle Doxorubicin AcBut-CM AcBut-CM AcBut-CM AcBut-CM
0 178 .+-. 18 166 .+-. 18 191 .+-. 41 173 .+-. 24 161 .+-. 21 174
.+-. 23 7 468 .+-. 87 494 .+-. 70 472 .+-. 31 461 .+-. 36 441 .+-.
57 428 .+-. 46 11 1138 .+-. 41 752 .+-. 92 1176 .+-. 9 467 .+-. 75
209 .+-. 42 290 .+-. 42 18 GT 966 .+-. 32 GT 239 .+-. 40 83 .+-. 25
69 .+-. 7 25 GT GT GT 137 .+-. 27 30 .+-. 8 34 .+-. 6 32 GT GT GT
254 .+-. 53 15 .+-. 10 3 .+-. 3 39 GT GT GT 485 .+-. 73 14 .+-. 9 0
.+-. 0 47 GT GT GT 903 .+-. 145 32 .+-. 25 0 .+-. 0 53 GT GT GT GT
82 .+-. 66 0 .+-. 0 60 GT GT GT GT 159 .+-. 118 0 .+-. 0 67 GT GT
GT GT 318 .+-. 209 0 .+-. 0 74 GT GT GT GT 452 .+-. 234 0 .+-. 0 81
GT GT GT GT 811 .+-. 362 0 .+-. 0 89 GT GT GT GT GT 0 .+-. 0 95 GT
GT GT GT GT 0 .+-. 0 102 GT GT GT GT GT 0 .+-. 0 109 GT GT GT GT GT
0 .+-. 0 116 GT GT GT GT GT 0 .+-. 0 123 GT GT GT GT GT 0 .+-. 0
130 GT GT GT GT GT 0 .+-. 0 137 GT GT GT GT GT 0 .+-. 0 144 GT GT
GT GT GT 0 .+-. 0 152 GT GT GT GT GT 0 .+-. 0 155 GT GT GT GT GT 0
.+-. 0 168 GT GT GT GT GT 0 .+-. 0 172 GT GT GT GT GT 0 .+-. 0
[0425] Table 17 and FIG. 12 show the efficacy of huE22-AcBut-CM and
huE47-AcBut-CM in the Breast-13 (BR13) TNBC PDX. For
huE22-AcBut-CM, the 0.27 mg/kg dose level produced regressions for
90 days after which the tumors regrew. Further, the 0.09 mg/kg dose
level delayed tumor growth for .about.40 days. In contrast, neither
the control ADC nor doxorubicin SOC impacted tumor growth.
TABLE-US-00017 TABLE 17 Efficacy of huE22-AcBut-CM and
huE47-AcBut-CM in BR13 TNBC PDX 0.3 0.036 0.09 0.27 0.1 0.3 mg/kg
mg/kg mg/kg mg/kg mg/kg mg/kg Control- huE22- huE22- huE22- huE47-
huE47- Day Vehicle Doxorubicin AcBut-CM AcBut-CM AcBut-CM AcBut-CM
AcBut-CM AcBut-CM 0 161 .+-. 15 167 .+-. 13 154 .+-. 11 156 .+-. 13
156 .+-. 10 155 .+-. 14 19 .+-. 25 172 .+-. 26 7 180 .+-. 15 178
.+-. 20 171 .+-. 20 176 .+-. 20 145 .+-. 14 144 .+-. 15 251 .+-. 42
242 .+-. 37 14 239 .+-. 27 227 .+-. 25 183 .+-. 12 184 .+-. 24 156
.+-. 21 91 .+-. 14 261 .+-. 42 238 .+-. 38 22 382 .+-. 52 322 .+-.
38 237 .+-. 24 232 .+-. 32 112 .+-. 20 35 .+-. 5 264 .+-. 47 92
.+-. 18 28 484 .+-. 72 326 .+-. 34 287 .+-. 28 279 .+-. 47 98 .+-.
23 17 .+-. 4 439 .+-. 86 34 .+-. 7 36 600 .+-. 98 GT 315 .+-. 30
341 .+-. 42 116 .+-. 32 4 .+-. 1 509 .+-. 105 41 .+-. 21 42 735
.+-. 106 GT 402 .+-. 19 471 .+-. 68 138 .+-. 34 3 .+-. 1 546 .+-.
121 46 .+-. 15 49 841 .+-. 106 GT 493 .+-. 38 516 .+-. 91 188 .+-.
46 1 .+-. 1 698 .+-. 144 79 .+-. 27 56 GT GT 659 .+-. 41 738 .+-.
109 274 .+-. 75 6 .+-. 2 713 .+-. 132 155 .+-. 57 63 GT GT 779 .+-.
33 814 .+-. 110 328 .+-. 86 7 .+-. 3 960 .+-. 192 135 .+-. 51 70 GT
GT 991 .+-. 71 959 .+-. 103 425 .+-. 93 13 .+-. 4 GT 167 .+-. 56 77
GT GT GT GT 531 .+-. 116 13 .+-. 7 GT 132 .+-. 46 84 GT GT GT GT
667 .+-. 133 30 .+-. 13 GT 151 .+-. 50 91 GT GT GT GT 830 .+-. 146
40 .+-. 15 GT 198 .+-. 62 98 GT GT GT GT GT 79 .+-. 33 GT 559 .+-.
186 105 GT GT GT GT GT 99 .+-. 36 GT 788 .+-. 257 112 GT GT GT GT
GT 147 .+-. 53 GT 744 .+-. 217 119 GT GT GT GT GT 198 .+-. 65 GT
1030 .+-. 349 126 GT GT GT GT GT 290 .+-. 97 GT GT 133 GT GT GT GT
GT 332 .+-. 113 GT GT 140 GT GT GT GT GT 522 .+-. 156 GT GT 144 GT
GT GT GT GT 573 .+-. 144 GT GT 150 GT GT GT GT GT 709 .+-. 248 GT
GT
[0426] Table 18 and FIG. 13 show the efficacy of huE22-AcBut-CM and
huE47-AcBut-CM in the Breast-22 (BR22) PDX, a less sensitive tumor
model of TNBC. For huE22-AcBut-CM, the 0.27 mg/kg dose level
delayed tumor growth for .about.40 days but did not produce any
complete regressions. Further, dose levels below 0.27 mg/kg had
minimal effect. Neither the control ADC nor doxorubicin SOC
impacted tumor growth.
TABLE-US-00018 TABLE 18 Efficacy of huE22-AcBut-CM and
huE47-AcBut-CM in BR22 TNBC PDX 0.1 0.036 0.09 0.27 0.3 mg/kg mg/kg
mg/kg mg/kg mg/kg Control- huE22- huE22- huE22- huE47- Day Vehicle
Doxorubicin AcBut-CM AcBut-CM AcBut-CM AcBut-CM AcBut-CM 0 195 .+-.
17 244 .+-. 17 208 .+-. 23 207 .+-. 17 217 .+-. 17 202 .+-. 16 194
.+-. 17 7 376 .+-. 43 416 .+-. 61 354 .+-. 35 358 .+-. 41 324 .+-.
52 292 .+-. 32 438 .+-. 36 9 401 .+-. 42 -- 376 .+-. 23 366 .+-. 37
319 .+-. 63 281 .+-. 28 522 .+-. 48 14 564 .+-. 54 553 .+-. 39 494
.+-. 24 511 .+-. 60 364 .+-. 45 248 .+-. 31 468 .+-. 58 21 819 .+-.
97 729 .+-. 91 677 .+-. 52 649 .+-. 83 368 .+-. 58 92 .+-. 15 557
.+-. 80 28 GT GT 1054 .+-. 122 891 .+-. 106 535 .+-. 70 77 .+-. 21
855 .+-. 112 35 GT GT GT GT 715 .+-. 90 81 .+-. 24 1145 .+-. 165 43
GT GT GT GT 1007 .+-. 115 140 .+-. 41 GT 49 GT GT GT GT GT 199 .+-.
53 GT 56 GT GT GT GT GT 274 .+-. 78 GT 63 GT GT GT GT GT 370 .+-.
73 GT 70 GT GT GT GT GT 605 .+-. 120 GT 77 GT GT GT GT GT 711 .+-.
110 GT 84 GT GT GT GT GT 981 .+-. 135 GT
[0427] FIG. 19 shows the efficacy of huE15, huE22 and huE47
conjugated to various io microtubule inhibitors (MTIs) in the BR22
PDX, such as vc0101 and mc8261. The data demonstrates that the MTI
ADCs did not significantly impact tumor growth. Therefore, it was
unexpected that huE22-AcBut-CM and huE47-AcBut-CM demonstrated
increased efficacy in the same BR22 PDX model.
[0428] Table 19 and FIG. 14 show the efficacy of huE22-AcBut-CM in
the Breast-31 (BR31) TNBC PDX. The 0.27 mg/kg dose level produced
regressions for .about.120 days, after which the tumors regrew in
some animals. The 0.09 mg/kg dose level regressed tumors for
.about.50 days, after which the tumors regrew. In contrast, neither
the control ADC nor doxorubicin SOC impacted tumor growth.
TABLE-US-00019 TABLE 19 Efficacy of huE22-AcBut-CM in BR31 TNBC PDX
0.3 0.09 0.27 0.1 0.3 mg/kg mg/kg mg/kg mg/kg mg/kg Control- huE22-
huE22- huE47- huE47- Day Vehicle Doxorubicin AcBut-CM AcBut-CM
AcBut-CM AcBut-CM AcBut-CM 0 159 .+-. 10 160 .+-. 11 163 .+-. 23
165 .+-. 6 149 .+-. 13 163 .+-. 6 147 .+-. 15 7 269 .+-. 17 304
.+-. 20 305 .+-. 42 277 .+-. 18 137 .+-. 11 283 .+-. 26 239 .+-. 27
14 425 .+-. 32 458 .+-. 24 307 .+-. 53 201 .+-. 11 59 .+-. 8 269
.+-. 29 141 .+-. 17 21 668 .+-. 50 592 .+-. 56 377 .+-. 58 103 .+-.
12 30 .+-. 9 127 .+-. 11 44 .+-. 11 28 1088 .+-. 93 759 .+-. 111
356 .+-. 52 64 .+-. 4 11 .+-. 7 126 .+-. 16 22 .+-. 8 36 GT GT 405
.+-. 52 60 .+-. 11 17 .+-. 9 179 .+-. 20 4 .+-. 4 44 GT GT 569 .+-.
64 57 .+-. 15 0 .+-. 0 213 .+-. 33 6 .+-. 6 49 GT GT 557 .+-. 73 82
.+-. 19 0 .+-. 0 373 .+-. 39 11 .+-. 7 56 GT GT 795 .+-. 88 132
.+-. 32 0 .+-. 0 750 .+-. 76 5 .+-. 5 63 GT GT 1160 .+-. 142 206
.+-. 44 0 .+-. 0 GT 9 .+-. 9 71 GT GT GT 338 .+-. 73 0 .+-. 0 GT 27
.+-. 27 78 GT GT GT 414 .+-. 74 0 .+-. 0 GT 75 .+-. 64 85 GT GT GT
589 .+-. 121 12 .+-. 8 GT 63 .+-. 56 91 GT GT GT 741 .+-. 157 20
.+-. 13 GT 117 .+-. 107 98 GT GT GT 1178 .+-. 271 26 .+-. 17 GT 154
.+-. 137 105 GT GT GT GT 32 .+-. 21 GT 165 .+-. 147 112 GT GT GT GT
29 .+-. 20 GT 205 .+-. 181 119 GT GT GT GT 58 .+-. 39 GT 210 .+-.
180 126 GT GT GT GT 88 .+-. 58 GT 267 .+-. 209 133 GT GT GT GT 159
.+-. 103 GT 286 .+-. 211 141 GT GT GT GT 181 .+-. 118 GT 286 .+-.
211 147 GT GT GT GT 219 .+-. 142 GT 286 .+-. 211 154 GT GT GT GT
279 .+-. 176 GT 428 .+-. 302 161 GT GT GT GT 293 .+-. 172 GT 571
.+-. 376 168 GT GT GT GT 293 .+-. 172 GT 571 .+-. 376 175 GT GT GT
GT 306 .+-. 170 GT GT 182 GT GT GT GT 308 .+-. 169 GT GT 189 GT GT
GT GT 317 .+-. 168 GT GT 197 GT GT GT GT 429 .+-. 192 GT GT 204 GT
GT GT GT 445 .+-. 200 GT GT
[0429] FIG. 20 shows the efficacy of huE15, huE22 and huE47
conjugated to the MTI vc0101 in the BR31 PDX. The data demonstrates
that the MTI ADCs did not significantly impact tumor growth.
Therefore, it was unexpected that huE22-AcBut-CM and huE47-AcBut-CM
demonstrated increased efficacy in the same BR22 PDX model.
[0430] Table 20 shows the efficacy of huE22-AcBut-CM in the
Breast-56 (BR56) TNBC PDX. In contrast, neither the control ADC nor
doxorubicin SOC impacted tumor growth.
TABLE-US-00020 TABLE 20 Efficacy of huE22-AcBut-CM in BR56 TNBC PDX
0.1 0.036 0.09 0.27 mg/kg mg/kg mg/kg mg/kg Control- huE22- huE22-
huE22- Day Vehicle Doxorubicin AcBut-CM AcBut-CM AcBut-CM AcBut-CM
0 144 .+-. 9 141 .+-. 13 140 .+-. 11 143 .+-. 13 148 .+-. 14 141
.+-. 12 4 179 .+-. 15 182 .+-. 11 181 .+-. 12 154 .+-. 13 150 .+-.
10 151 .+-. 14 11 271 .+-. 33 267 .+-. 31 270 .+-. 26 268 .+-. 42
268 .+-. 32 170 .+-. 21 18 373 .+-. 33 318 .+-. 32 408 .+-. 34 298
.+-. 55 251 .+-. 25 90 .+-. 10 25 727 .+-. 86 374 .+-. 50 644 .+-.
49 424 .+-. 89 286 .+-. 39 42 .+-. 13 33 1010 .+-. 88 GT 913 .+-.
84 431 .+-. 98 291 .+-. 45 14 .+-. 4 40 GT GT GT 591 .+-. 120 341
.+-. 47 0 .+-. 0 47 GT GT GT 635 .+-. 116 442 .+-. 66 0 .+-. 0 54
GT GT GT 780 .+-. 125 635 .+-. 94 11 .+-. 8 60 GT GT GT 896 .+-.
132 816 .+-. 64 48 .+-. 17 67 GT GT GT GT GT 75 .+-. 29 74 GT GT GT
GT GT 155 .+-. 51 81 GT GT GT GT GT 207 .+-. 56 88 GT GT GT GT GT
270 .+-. 78 95 GT GT GT GT GT 363 .+-. 100 102 GT GT GT GT GT 530
.+-. 143 109 GT GT GT GT GT 714 .+-. 198
B. Ovarian Cancer
[0431] Table 21 and FIG. 15 show the efficacy of huE22-AcBut-CM in
the Ovarian-45 (OV45) ovarian cancer PDX. Sustained regressions for
more than 75 days were achieved with 0.27 mg/kg. Regressions were
also achieved at the 0.09 mg/kg dose level, with tumor regrowth in
some animals after 50 days. The 0.036 dose level delayed tumor
growth for .about.25 days. In contrast the control ADC did not
impact tumor growth.
TABLE-US-00021 TABLE 21 Efficacy of huE22-AcBut-CM in OV45 Ovarian
Cancer PDX 0.1 mg/kg 0.036 mg/kg 0.09 mg/kg 0.27 mg/kg 10 mg/kg
Control- huE22- huE22- huE22- Day Mouse IgG1 AcBut-CM AcBut-CM
AcBut-CM AcBut-CM 0 157 .+-. 16 142 .+-. 14 141 .+-. 14 145 .+-. 15
153 .+-. 20 3 -- 239 .+-. 35 178 .+-. 5 240 .+-. 41 177 .+-. 39 8
329 .+-. 32 470 .+-. 77 198 .+-. 17 147 .+-. 23 35 .+-. 5 14 516
.+-. 57 590 .+-. 101 206 .+-. 34 60 .+-. 16 4 .+-. 2 21 843 .+-.
151 906 .+-. 194 251 .+-. 53 15 .+-. 1 0 .+-. 0 33 GT GT 429 .+-.
79 0 .+-. 0 0 .+-. 0 39 GT GT 733 .+-. 111 0 .+-. 0 0 .+-. 0 46 GT
GT 1140 .+-. 92 0 .+-. 0 0 .+-. 0 53 GT GT GT 30 .+-. 30 0 .+-. 0
60 GT GT GT 118 .+-. 99 0 .+-. 0 67 GT GT GT 256 .+-. 165 0 .+-. 0
74 GT GT GT 423 .+-. 257 0 .+-. 0 81 GT GT GT 443 .+-. 252 0 .+-. 0
88 GT GT GT 743 .+-. 256 0 .+-. 0 96 GT GT GT 1576 .+-. 208 0 .+-.
0 102 GT GT GT GT 0 .+-. 0 109 GT GT GT GT 0 .+-. 0 116 GT GT GT GT
0 .+-. 0 123 GT GT GT GT 0 .+-. 0 131 GT GT GT GT 0 .+-. 0 137 GT
GT GT GT 0 .+-. 0 141 GT GT GT GT 0 .+-. 0 158 GT GT GT GT 0 .+-. 0
166 GT GT GT GT 0 .+-. 0
[0432] Table 22 and FIG. 16 show the efficacy of huE22-AcBut-CM in
the Ovarian-55 (OV55) ovarian cancer PDX. Tumor regressions were
achieved at the 0.27 and 0.09 mg/kg dose levels, with the 0.036
dose level producing some delay in tumor growth. The control ADC
did not impact tumor growth.
TABLE-US-00022 TABLE 22 Efficacy of huE22-AcBut-CM in OV55 Ovarian
Cancer PDX 0.1 mg/kg 0.036 mg/kg 0.09 mg/kg 0.27 mg/kg Control-
huE22- huE22- huE22- Day Vehicle AcBut-CM AcBut-CM AcBut-CM
AcBut-CM 0 206 .+-. 41 216 .+-. 31 211 .+-. 28 208 .+-. 24 204 .+-.
28 6 317 .+-. 42 325 .+-. 57 262 .+-. 36 198 .+-. 33 152 .+-. 18 13
508 .+-. 45 412 .+-. 92 250 .+-. 46 109 .+-. 27 66 .+-. 13 20 717
.+-. 102 595 .+-. 111 232 .+-. 29 63 .+-. 14 35 .+-. 9 27 1064 .+-.
144 799 .+-. 173 355 .+-. 55 30 .+-. 9 5 .+-. 4 34 GT 1097 .+-. 123
630 .+-. 93 37 .+-. 13 0 .+-. 0 42 GT GT 863 .+-. 41 94 .+-. 37 0
.+-. 0 48 GT GT GT 171 .+-. 62 0 .+-. 0 55 GT GT GT 382 .+-. 142 0
.+-. 0 62 GT GT GT 579 .+-. 209 4 .+-. 4 69 GT GT GT 980 .+-. 405 7
.+-. 7 76 GT GT GT GT 11 .+-. 11 83 GT GT GT GT 24 .+-. 24 90 GT GT
GT GT 35 .+-. 35 97 GT GT GT GT 78 .+-. 78 105 GT GT GT GT 107 .+-.
107 108 GT GT GT GT 99 .+-. 99 121 GT GT GT GT 157 .+-. 157 125 GT
GT GT GT 253 .+-. 253 132 GT GT GT GT 253 .+-. 253
[0433] Table 23 and FIG.17 show the efficacy of huE22-AcBut-CM in
the Ovarian-44 (OV44) ovarian cancer PDX, a less sensitive tumor
model of ovarian cancer. The 0.27 mg/kg dose level delayed tumor
growth for .about.50 days but did not regress the tumors. Dose
levels below 0.27 mg/kg had minimal effect. The control ADC did not
impact tumor growth.
TABLE-US-00023 TABLE 23 Efficacy of huE22-AcBut-CM in OV44 Ovarian
Cancer PDX 0.1 mg/kg 0.036 mg/kg 0.09 mg/kg 0.27 mg/kg Control-
huE22- huE22- huE22- Day Vehicle AcBut-CM AcBut-CM AcBut-CM
AcBut-CM 0 141 .+-. 14 138 .+-. 18 140 .+-. 9 148 .+-. 16 152 .+-.
17 5 286 .+-. 58 215 .+-. 30 269 .+-. 33 236 .+-. 20 249 .+-. 35 11
442 .+-. 27 347 .+-. 59 331 .+-. 45 328 .+-. 39 250 .+-. 30 18 555
.+-. 14 417 .+-. 72 328 .+-. 53 299 .+-. 29 162 .+-. 24 25 747 .+-.
35 621 .+-. 94 397 .+-. 59 313 .+-. 39 142 .+-. 21 32 1010 .+-. 120
760 .+-. 104 478 .+-. 69 414 .+-. 50 137 .+-. 33 39 GT 851 .+-. 68
567 .+-. 79 497 .+-. 44 114 .+-. 43 46 GT GT 708 .+-. 61 610 .+-.
28 140 .+-. 59 53 GT GT 1060 .+-. 182 957 .+-. 99 249 .+-. 152 61
GT GT GT GT 242 .+-. 102 67 GT GT GT GT 367 .+-. 143 74 GT GT GT GT
452 .+-. 140 81 GT GT GT GT 521 .+-. 130 88 GT GT GT GT 619 .+-.
120 95 GT GT GT GT 686 .+-. 111 102 GT GT GT GT 830 .+-. 82
[0434] Table 24 and FIG. 18 show the efficacy of huE22-AcBut-CM in
the Ovarian-63 (OV63) ovarian cancer PDX, a less sensitive model of
ovarian cancer. Transient tumor regressions were achieved at 0.27
mg/kg and delays in tumor growth achieved at 0.036 and 0.09
mg/kg.
TABLE-US-00024 TABLE 24 Efficacy of huE22-AcBut-CM in OV63 Ovarian
Cancer PDX 0.1 0.036 0.09 0.27 5.0 mg/kg mg/kg mg/kg mg/kg mg/kg
Control- huE22- huE22- huE22- Day Vehicle Cisplatin AcBut-CM
AcBut-CM AcBut-CM AcBut-CM 0 160 .+-. 22 153 .+-. 22 155 .+-. 18
153 .+-. 21 156 .+-. 16 165 .+-. 16 5 152 .+-. 10 146 .+-. 27 212
.+-. 30 197 .+-. 25 176 .+-. 30 158 .+-. 15 11 171 .+-. 23 -- 178
.+-. 23 160 .+-. 16 180 .+-. 38 112 .+-. 19 18 207 .+-. 16 198 .+-.
29 221 .+-. 40 209 .+-. 33 152 .+-. 36 86 .+-. 15 25 218 .+-. 37
205 .+-. 36 277 .+-. 36 178 .+-. 17 112 .+-. 17 67 .+-. 18 32 207
.+-. 17 238 .+-. 49 316 .+-. 32 219 .+-. 37 132 .+-. 29 83 .+-. 22
39 216 .+-. 10 221 .+-. 40 381 .+-. 21 264 .+-. 72 184 .+-. 24 106
.+-. 44 47 249 .+-. 29 243 .+-. 42 533 .+-. 80 271 .+-. 73 186 .+-.
16 104 .+-. 40 53 317 .+-. 59 265 .+-. 63 504 .+-. 79 322 .+-. 108
188 .+-. 31 78 .+-. 30 60 317 .+-. 63 296 .+-. 80 643 .+-. 116 326
.+-. 110 179 .+-. 28 60 .+-. 43 67 446 .+-. 123 265 .+-. 82 805
.+-. 165 417 .+-. 141 184 .+-. 27 69 .+-. 38 74 ND ND 840 .+-. 187
456 .+-. 190 209 .+-. 46 73 .+-. 45 81 ND ND GT 498 .+-. 188 241
.+-. 66 65 .+-. 39 88 ND ND GT 530 .+-. 191 251 .+-. 3 64 .+-.
26
[0435] Table 25 shows the efficacy of huE22-AcBut-CM in the
Ovarian-39 (OV39) ovarian cancer PDX.
TABLE-US-00025 TABLE 25 Efficacy of huE22-AcBut-CM in OV39 Ovarian
Cancer PDX 0.1 0.036 0.09 0.27 mg/kg mg/kg mg/kg mg/kg Control-
huE22- huE22- huE22- Day Vehicle Doxorubicin AcBut-CM AcBut-CM
AcBut-CM AcBut-CM 0 177 .+-. 32 186 .+-. 33 163 .+-. 23 165 .+-. 29
173 .+-. 18 169 .+-. 21 7 413 .+-. 103 282 .+-. 50 413 .+-. 74 448
.+-. 78 355 .+-. 48 378 .+-. 56 14 806 .+-. 333 171 .+-. 32 650
.+-. 118 814 .+-. 112 554 .+-. 80 320 .+-. 73 17 1101 .+-. 502 122
.+-. 24 822 .+-. 153 1050 .+-. 159 609 .+-. 94 252 .+-. 62 21 1305
.+-. 453 106 .+-. 19 891 .+-. 143 1265 .+-. 146 628 .+-. 97 147
.+-. 28 24 1436 .+-. 427 83 .+-. 11 1096 .+-. 170 1356 .+-. 185 614
.+-. 95 118 .+-. 28 28 GT 118 .+-. 17 1245 .+-. 154 GT 779 .+-. 123
98 .+-. 25 35 GT 361 .+-. 40 GT GT 934 .+-. 99 67 .+-. 18 42 GT 712
.+-. 97 GT GT GT 72 .+-. 23 49 GT 966 .+-. 85 GT GT GT 170 .+-. 96
56 GT GT GT GT GT 446 .+-. 337 63 GT GT GT GT GT 940 .+-. 675 70 GT
GT GT GT GT 1194 .+-. 422
C. Small-Cell Lung Cancer (SCLC) and Colorectal Cancer
[0436] Several additional efficacy studies were performed in models
of small-cell lung cancer (SCLC), LU80 and LU86 PDXs, and
colorectal cancer (CRC), CR5 PDX, with data shown in Tables 26-28.
The results obtained were unexpected because huE22-ActBut-CM
demonstrated superior efficacy in the SCLC and CRC PDX models
compared to the treatment with doxorubicin standard-of-care (SOC)
which has a similar mechanism of action to calicheamicin (DNA
damaging agent).
TABLE-US-00026 TABLE 26 Efficacy of huE22-AcBut-CM in LU80 SCLC PDX
5.0 mg/kg 0.1 0.018 0.036 0.09 0.27 Cisplatin + mg/kg mg/kg mg/kg
mg/kg mg/kg 24 mg/kg Control- huE22- huE22- huE22- huE22- Day
Vehicle Etoposide AcBut-CM AcBut-CM AcBut-CM AcBut-CM AcBut-CM 0
165 .+-. 27 190 .+-. 8 188 .+-. 19 158 .+-. 29 177 .+-. 19 161 .+-.
18 158 .+-. 20 7 500 .+-. 64 89 .+-. 11 312 .+-. 17 511 .+-. 37 527
.+-. 64 384 .+-. 40 208 .+-. 36 14 824 .+-. 137 163 .+-. 31 754
.+-. 41 630 .+-. 79 452 .+-. 75 217 .+-. 31 80 .+-. 10 21 985 .+-.
116 325 .+-. 68 1026 .+-. 61 638 .+-. 58 464 .+-. 67 134 .+-. 20 49
.+-. 8 28 GT 616 .+-. 67 GT 647 .+-. 96 439 .+-. 75 83 .+-. 24 7
.+-. 5 35 GT 979 .+-. 135 GT 1324 .+-. 208 883 .+-. 174 112 .+-. 31
0 .+-. 0 42 GT GT GT GT GT 126 .+-. 38 0 .+-. 0 49 GT GT GT GT GT
330 .+-. 120 0 .+-. 0 56 GT GT GT GT GT 686 .+-. 249 46 .+-. 31 63
GT GT GT GT GT 979 .+-. 216 105 .+-. 52 70 GT GT GT GT GT GT 227
.+-. 89 77 GT GT GT GT GT GT 376 .+-. 145 84 GT GT GT GT GT GT 621
.+-. 209 91 GT GT GT GT GT GT 703 .+-. 225
TABLE-US-00027 TABLE 27 Efficacy of huE22-AcBut-CM and
huE47-AcBut-CM in LU86 SCLC PDX 5.0 mg/kg 0.3 0.036 0.09 0.27 0.1
0.3 Cisplatin + mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 24 mg/kg
Control- huE22- huE22- huE22- huE47- huE47- Day Vehicle Etoposide
AcBut-CM AcBut-CM AcBut-CM AcBut-CM AcBut-CM AcBut-CM 0 147 .+-. 11
149 .+-. 13 205 .+-. 19 139 .+-. 9 143 .+-. 10 138 .+-. 9 161 .+-.
16 195 .+-. 17 7 317 .+-. 33 250 .+-. 24 282 .+-. 24 239 .+-. 25
215 .+-. 19 105 .+-. 12 234 .+-. 24 372 .+-. 60 14 672 .+-. 62 468
.+-. 47 458 .+-. 62 296 .+-. 33 66 .+-. 9 0 .+-. 0 231 .+-. 34 260
.+-. 65 21 1233 .+-. 83 946 .+-. 77 637 .+-. 90 423 .+-. 50 0 .+-.
0 0 .+-. 0 355 .+-. 58 212 .+-. 84 28 GT GT 964 .+-. 130 557 .+-.
66 0 .+-. 0 0 .+-. 0 458 .+-. 79 304 .+-. 111 35 GT GT GT 808 .+-.
69 0 .+-. 0 0 .+-. 0 598 .+-. 94 433 .+-. 162 42 GT GT GT GT 0 .+-.
0 0 .+-. 0 808 .+-. 113 585 .+-. 198 49 GT GT GT GT 0 .+-. 0 0 .+-.
0 1015 .+-. 139 778 .+-. 258 56 GT GT GT GT 0 .+-. 0 0 .+-. 0 1262
.+-. 198 1031 .+-. 304 63 GT GT GT GT 28 .+-. 22 0 .+-. 0 1770 .+-.
293 GT 70 GT GT GT GT 52 .+-. 34 0 .+-. 0 GT GT 77 GT GT GT GT 93
.+-. 60 0 .+-. 0 GT GT 84 GT GT GT GT 117 .+-. 75 0 .+-. 0 GT GT 92
GT GT GT GT 237 .+-. 124 6 .+-. 6 GT GT 98 GT GT GT GT 304 .+-. 154
34 .+-. 24 GT GT 105 GT GT GT GT 385 .+-. 179 56 .+-. 32 GT GT 112
GT GT GT GT 420 .+-. 171 99 .+-. 56 GT GT 119 GT GT GT GT 491 .+-.
163 171 .+-. 109 GT GT 126 GT GT GT GT 602 .+-. 165 239 .+-. 139 GT
GT 133 GT GT GT GT 828 .+-. 213 370 .+-. 201 GT GT 140 GT GT GT GT
GT 429 .+-. 210 GT GT 147 GT GT GT GT GT 469 .+-. 222 GT GT 154 GT
GT GT GT GT 599 .+-. 286 GT GT
TABLE-US-00028 TABLE 28 Efficacy of huE22-AcBut-CM in CR5
Colorectal Cancer PDX 0.1 0.3 mg/kg 0.09 0.27 0.3 10 35 mg/kg
Control mg/kg mg/kg mg/kg mg/kg mg/kg Control- hlgGl- huE22- huE22-
huE47- Day MslgG1 Irinotecan AcBut-CM AcBut-CM AcBut-CM AcBut-CM
AcBut-CM 0 249 .+-. 17 252 .+-. 15 141 .+-. 14 165 .+-. 10 168 .+-.
9 141 .+-. 9 141 .+-. 7 3 397 .+-. 35 395 .+-. 29 234 .+-. 22 246
.+-. 23 255 .+-. 15 210 .+-. 11 223 .+-. 10 7 344 .+-. 32 237 .+-.
23 357 .+-. 30 166 .+-. 23 154 .+-. 11 299 .+-. 18 361 .+-. 20 10
829 .+-. 64 446 .+-. 35 524 .+-. 49 502 .+-. 59 429 .+-. 37 286
.+-. 15 498 .+-. 25 14 1250 .+-. 107 448 .+-. 38 376 .+-. 41 831
.+-. 88 531 .+-. 35 93 .+-. 5 329 .+-. 17 21 GT 396 .+-. 52 1150
.+-. 75 1406 .+-. 123 763 .+-. 64 161 .+-. 10 885 .+-. 51 28 GT 629
.+-. 116 GT 2372 .+-. 165 1457 .+-. 90 149 .+-. 17 1273 .+-. 81 35
GT 1097 .+-. 142 GT GT GT 397 .+-. 90 1724 .+-. 103 42 GT GT GT GT
GT 889 .+-. 171 GT
[0437] The results of all in vivo efficacy studies with
huE22-AcBut-CM are summarized in Tables 29 and 30. In summary,
huE22-AcBut-CM strongly inhibited tumor growth in models of TNBC
(non-Claudin low), ovarian cancer, SCLC and colorectal cancer.
huE22-AcBut-CM outperformed standard-of-care chemotherapy (SOC) in
all cases tested (TNBC and SCLC). Tumor regression was defined as
reduction in mean tumor volume relative to the size at first
dose.
[0438] In breast and ovarian models, tumor regression was defined
as a reduction in mean tumor volume after dosing. TGI=Tumor Growth
Inhibition. % TGI=[1-(Mean Tumor Volume of Treated)/(Mean Tumor
Volume of Vehicle)]. In cases where tumors regressed, Time To
Progression (TTP) was determined to be the number of days between
the first dose and the time at which mean tumor volume
significantly increased (regrew) after regression. If the tumor did
not regrow during the course of the experiment, TTP is the number
of days between the first dose and the end of the experiment. As
described above, all animals were dosed twice a week for 4 cycles
with huE22-AcBut-CM ADC, once a week for 2 cycles with doxorubicin
at 1.5 mg/kg or once a week for 2 cycles with cisplatin at 5 mg/kg.
ADC dose levels are listed in mg/kg according to antibody content.
Animals were dosed intraperitoneally in all studies except 144580,
in which they were dosed intravenously.
TABLE-US-00029 TABLE 29 Summary of huE22-AcBut-CM breast cancer in
vivo efficacy studies 1.5 mpk Doxorubicin huE22-AcBut-CM Breast
Regression EFNA4 Dose Regression Cancer Tumor (TTP, Days) Protein
Level (TTP, Days) Subtype model or % TGI (ng/mg) (mg/kg) or % TGI
TNBC: BR5 44% TGI 1.8 0.036 Regression (32) Basal PDX 0.09
Regression (53) 0.27 Regression (200) BR22 27% TGI 1.91 0.018 No
activity PDX 0.036 21% TGI 0.09 55% TGI 0.27 Regression (43) BR31
30% TGI 1.02 0.09 Regression (56) PDX 0.27 Regression (91) BR56 49%
TGI 1.01 0.018 No activity PDX 0.036 34% TGI 0.09 56% TGI 0.27
Regression (60) TNBC: BR13 16% TGI 1.00 0.018 No activity NL 0.036
36% TGI 0.09 Regression (42) 0.27 Regression (84) TNBC: 144580 ND
1.74 0.03 11% TGI Unknown 0.1 55% TGI 0.3 Regression (53) TNBC:
BR25 No activity 0.47 0.27 27% TGI CL BR64 No activity 0.66 0.27 No
activity HER2+ BR17 66% TGI ND 0.27 No Activity NL = Normal-like.
CL = Claudin-low. ND = no determined.
TABLE-US-00030 TABLE 30 Summary of huE22-AcBut-CM ovarian cancer in
vivo efficacy studies 5 mpk Cisplatin huE22-AcBut-CM Ovarian
Regression EFNA4 Dose Regression Cancer Tumor (TTP, Days) Protein
Level (TTP, Days) Subtype model or % TGI (ng/mg) (mg/kg) or % TGI
High OV39 Regression (28) ND 0.036 No activity Grade 0.09 45% TGI
Serous 0.27 Regression (49) OV44 Regression (60) 1.39 0.036 53% TGI
PDX 0.09 59% TGI 0.27 Regression (53) OV63 35% TGI 0.49 0.036 No
activity PDX 0.09 59% TGI 0.27 Regression (88) MMMT OV45 NA 3.64
0.018 No activity PDX 0.036 70% TGI 0.09 Regression (60) 0.27
Regression (200) OV55 NA 0.42 0.018 No activity PDX 0.036 67% TGI
0.09 Regression (42) 0.27 Regression (76) OV124 NA ND 0.27
Regression (152) MMMT = malignant mixed Mullerian tumor; ND = not
determined; NA = not applicable.
Example 9
Reduction of Tumor-Initiating Cells (TIC)
[0439] To determine whether anti-EFNA4 antibody-drug conjugate
treatments reduced tumor-initiating cell (TIC) frequency in tumors,
tumors were pre-treated with anti-EFNA4 antibody-drug conjugate,
huE22-AcBut-CM, or control hIgG antibody-drug conjugate,
control-AcBut-CM, and then live human tumor cells from pre-treated
dissociated tumors were implanted into naive animals in a limit
dilution analysis. Tumors from huE22-AcBut-CM or control-AcBut-CM
treated tumor-bearing mice were harvested at day 12 (for BR22) and
day 21 (for BR13) post-initial treatment. The day of harvest serial
transplantation was chosen based on when tumors were starting to
regress following huE22-AcBut-CM exposure. Tumors were dissociated
and stained with anti-human ESA, anti-mouse CD45, and anti-mouse
H2Kd antibodies. Three tumors per treatment group were pooled, and
live human tumor cells (ESA+) were sorted by flow cytometry.
[0440] For BR22, groups of eight mice were injected with 400, 100,
50, or 20 tumor cells sorted from control-AcBut-CM treated tumors,
and groups of eight mice were injected with 390, 90, 40, or 18
tumor cells sorted from huE22-AcBut-CM treated tumors. For BR13,
groups of 10 mice were injected with 400, 100, 60, or 20 tumor
cells sorted from control-AcBut-CM treated tumors, and groups of 10
mice were injected with 380, 160, 50, or 25 tumor cells sorted from
huE22-AcBut-CM treated tumors.
[0441] Tumors in recipient mice were measured weekly and at 21
weeks post-injection, tumors that exceeded 200 mm.sup.3 in
recipient mice were scored as positive. Using Poisson distribution
statistics, via L-Calc software (Stemcell Technologies, Vancouver,
BC), injected cell doses of recipients with and without tumors at
21 weeks post-transplant were used to calculate the frequencies of
TIC in each population. For both the BR22 and BR13 PDX, the data
demonstrates anti-tumor efficacy using a secondary endpoint of TIC
frequencies, which is independent of standard tumor volume
measurements. The data further demonstrates huE22-AcBut-CM targets
the more aggressive/tumorgenic TICs (or CSCs).
[0442] The number of TIC in BR22 treated tumors were reduced by
about 3-fold from 1 TIC in 55 cells of control-AcBut-CM treated to
1 TIC in 147 cells of huE22-AcBut-CM treated (p=0.019 in two-tailed
test), as show in Table 31.
TABLE-US-00031 TABLE 31 Tumor-initiating cell frequency in BR22
tumor model. # Cells # Animals implanted with # Animals TIC Group
Pre-treatment per animal tumors in group frequency p-value A1
control-AcBut-CM 400 8 8 1 in 55 0.019 A2 control-AcBut-CM 100 6 8
A3 control-AcBut-CM 50 3 8 A4 control-AcBut-CM 20 6 8 B1
huE22-AcBut-CM 390 7 8 1 in 147 B2 huE22-AcBut-CM 90 6 8 B3
huE22-AcBut-CM 40 1 8 B4 huE22-AcBut-CM 18 0 7
[0443] Similarly, the number of TIC in BR13 treated tumors were
reduced by about 2.6-fold from 1 TIC in 75 cells of
control-AcBut-CM treated to 1 TIC in 270 cells of huE22-AcBut-CM
treated (p=0.0007), as shown in Table 32. In summary, mice injected
with huE22-AcBut-CM treated tumor cells consistently produced less
tumors than mice injected with similar number of control-AcBut-CM
treated tumor cells, indicating huE22-AcBut-CM treatment reduced
tumor-initiating cells (TIC).
TABLE-US-00032 TABLE 32 Tumor-initiating cell frequency in BR13
tumor model. # Cells # Animals implanted with # Animals TIC Group
Pre-treatment per animal tumors in group frequency p-value A1
control-AcBut-CM 400 10 10 1 in 75 0.0007 A2 control-AcBut-CM 100 7
10 A3 control-AcBut-CM 60 6 10 A4 control-AcBut-CM 20 2 10 B1
huE22-AcBut-CM 380 7 10 1 in 270 B2 huE22-AcBut-CM 160 4 10 B3
huE22-AcBut-CM 50 3 10 B4 huE22-AcBut-CM 25 1 9
Example 10
In Vitro Stability of the Total huE22-AcBut-CM in Plasma
[0444] Quantitation of the total ADC and total antibody, in plasma
samples from pharmacokinetic studies in mice dosed with the ADC was
determined using an enzyme-linked immunosorbent assay (ELISA)
method. ELISA and liquid chromatography tandem mass spectrometry
(LC-MS/MS) methods were also used to quantitate total ADC, total
antibody and unconjugated payload from the exploratory toxicology
studies in rat and monkey and in the in vitro plasma stability
study with the ADC.
[0445] The in vitro stability of the huE22-AcBut-CM, prepared
according the conjugation and purification processes described in
Example 6, was determined by measuring the total ADC and total
antibody by ELISA in Sprague Dawley rat and cynomolgus monkey
plasma. huE22-AcBut-CM was incubated at concentrations of 1 and 50
.mu.g/mL at 37.degree. C. up to 168 hours in mouse, rat, monkey,
and human plasma and the total ADC and total antibody (with the
exception of human plasma) were determined using an ELISA, see
Tables 33 and 34.
[0446] The mean (n=3) amounts of total antibody remaining after
incubation of huE22-AcBut-CM for 168 hours at 37.degree. C. in rat,
mouse, and monkey plasma were similar to the buffer control and
similar across species at both incubation concentrations. Mean
(n=3) total ADC and total antibody amounts remaining after
incubation of huE22-AcBut-CM in phosphate buffered saline (PBS)
w/1% bovine serum albumin (BSA) for 168 hours at 37.degree. C. were
94.5% and 90.5% respectively for the 1 .mu.g/mL incubation and 88.3
and 98.6% respectively for the 50 pg/mL incubation, indicating the
presence of minimal thermal degradation of the total ADC in plasma
at biological temperatures.
[0447] Total antibody concentrations could not be determined for
the total ADC incubations in human plasma due to ELISA limitations
of detection for a human IgG, in human plasma containing human IgG.
The mean amounts of total ADC remaining after incubation of
huE22-AcBut-CM for 168 hours at 37.degree. C. in rat, mouse,
monkey, and human plasma were similar to the buffer control and
similar across species at both incubation concentrations. NC=Not
calculated; SD=Standard deviation.
TABLE-US-00033 TABLE 33 % total huE22 Ab remaining in mouse, rat
and monkey plasma. Conc. Time (hours) Matrix (ug/mL) 0 8 24 48 72
168 Mouse 50 Mean 100 111 117 97.1 96.7 89.9 SD NC 25.4 16.3 18.1
20.8 24.0 1 Mean 100 105 126 109 101 102 SD NC 8.0 16.7 13.2 9.9
17.7 Rat 50 Mean 100 98.1 115 111 87.1 83.3 SD NC 8.0 21.0 21.0 3.7
13.3 1 Mean 100 76.8 89.0 81.4 79.4 64.6 SD NC 6.6 7.5 3.2 5.6 10.5
Monkey 50 Mean 100 106 109 106 103 94.3 SD NC 6.6 1.3 15.6 1.8 11.7
1 Mean 100 107 111 107 106 95.0 SD NC 8.9 2.0 15.9 5.7 12.0 Buffer
50 Mean 100 99.5 113 111 92.9 98.6 SD NC 8.7 5.2 11.2 10.4 13.5 1
Mean 100 86.1 87.5 91.9 92.1 90.5 SD NC 16.6 6.6 17.6 17.7 1.7
TABLE-US-00034 TABLE 34 % huE22-AcBut-CM remaining in mouse, rat,
monkey and human plasma Conc. Time (hours) Matrix (ug/mL) 0 8 24 48
72 168 Mouse 50 Mean 100 100 104.2 95.2 84.0 83.0 SD NC 13.7 11.7
11.3 10.0 3.6 1 Mean 100 96.7 101 102 87.5 79.1 SD NC 5.1 11.8 23.7
12.8 9.5 Rat 50 Mean 100 94.7 97.3 107.6 96.9 70.1 SD NC 1.6 6.7
21.7 7.9 10.6 1 Mean 100 95.2 93.0 86.2 81.0 69.0 SD NC 4.3 8.6 9.1
4.2 7.3 Monkey 50 Mean 100 100 103 87.9 81.9 71.6 SD NC 8.6 7.0 5.1
2.6 5.8 1 Mean 100 98.3 99.8 91.0 90.8 75.1 SD NC 8.7 13.1 6.5 5.3
2.5 Human 50 Mean 100 101 105 93.4 83.5 82.9 SD NC 15.1 12.0 13.8
11.5 3.4 1 Mean 100 102 93.2 98.4 89.3 82.5 SD NC 10.1 7.1 11.5 8.3
5.1 Buffer 50 Mean 100 90.6 104 99.7 93.4 88.3 SD NC 5.2 9.5 10.6
13.7 4.4 1 Mean 100 90.6 101 97.5 94.9 94.5 SD NC 7.5 17.4 4.5 3.0
2.8
Example 11
Mechanism of Action
[0448] The mechanism of action of huE22-AcBut-CM was analyzed to
demonstrate that it was consistent with that of calicheamicin.
Phosphorylated histone variant H2A.X (.gamma.H2A.X) is an
established biomarker of DNA damage. To validate the assay, cancer
cell lines were treated with unconjugated AcBut-CM and the
.gamma.H2A.X marker was evident in the cell nuclei in discrete
foci, which is the typical staining pattern. Treatment with
huE22-AcBut-CM resulted in DNA double-strand breaks in the target
cells both in vitro and in vivo, consistent with the expected
mechanism of action of a calicheamicin ADC.
[0449] HEK293T-EFNA4 or parental HEK293T cells were exposed for
four hours with 0.3 .mu.g/mL huE22-AcBut-CM, control ADC or
unconjugated huE22. Cells were washed, fixed with 4%
paraformaldehyde, permeabilized with 1% Triton-X100, incubated for
1 hour with anti-histone .gamma.H2A.X (Millipore #05-636), washed
and incubated for 30 minutes with AlexaFluor488-conjugated
secondary antibody and with DAPI nucleic acid stain, and then
washed and protected with mounting medium and coverslip. Cells were
visualized with a Zeiss LSM510 confocal microscope and analyzed for
the .gamma.H2A.X biomarker of DNA damage and DNA content.
[0450] After exposure to huE22-AcBut-CM, HEK293T-EFNA4 cells
exhibited discrete .gamma.H2A.X foci indicative of DNA damage; in
contrast no foci were observed after treatment with control ADC or
unconjugated huE22 (image not shown). Furthermore, huE22-AcBut-CM
did not induce foci in target-negative HEK293T cells. Thus,
huE22-AcBut-CM generated DNA damage in a target-dependent and
calicheamicin-dependent manner, consistent with the expected
mechanism of action of the ADC.
[0451] Analogous results were obtained in a pharmacodynamics study
in vivo. Mice that harbored EFNA4-expressing BR5 TNBC PDX tumors
were administered one dose of 1 mg/kg huE22-AcBut-CM, and tumors
were harvested after 24, 48 and 96 hours. Tumors were fixed in
formalin and embedded in paraffin (FFPE). Sections were stained
with 2.4 .mu.g/mL anti-hulgG (Cell Signaling #3443-1) and 1.8 mg/mL
anti-.gamma.-H2A.X (Cell Signaling 2577S) and slides were scanned
on an Aperio AT2. For digital image analysis, the region of healthy
viable tissue was classified while necrotic regions and irrelevant
tissues were eliminated. Individual cells within the viable region
were scored based on user-defined parameters (e.g. membrane hIgG
staining or intracellular .gamma.H2A.X staining), and the
percentage of marker-positive cells in the viable region was
reported.
[0452] As shown in FIG. 22, immunohistochemistry with anti-hIgG1
antibody (diagonal-hatched circles) demonstrated staining at the
plasma membrane of nearly every tumor cell at 24 hours after the
dose, and about half of the tumor cells at 96 hours. Further shown
in FIG. 22, immunohistochemistry with anti-.gamma.-H2A.X antibody
(open diamonds) revealed nuclear staining in tumor cells; as
expected, the time of peak anti-.gamma.-H2A.X staining lagged
behind the time of peak cell binding, since ADC internalization and
payload release occurs before the calicheamicin generates DNA
damage. The dashes indicate the median value per group. Together
the in vitro and in vivo studies demonstrated the expected
mechanism of action for anti-EFNA4-AcBut-CM ADCs.
[0453] Calicheamicin-generated DNA damage induces apoptosis, which
ultimately leads to cell death (Zein et al, 1988; Nicolaou et al,
1994; Prokop et al, 2003). Apoptosis following treatment with
huE22-AcBut-CM was evaluated by staining with Annexin V, which
marks apoptotic cells by binding to phosphatidylserine on the cell
surface (Koopman et al, 1994). HEK293T-EFNA4 or parental HEK293T
were treated with huE22-AcBut-CM, control ADC or huE22 mAb and then
stained with Annexin V and the viability stain 7AAD. Treatment of
HEK293T-EFNA4 cells with huE22-AcBut-CM resulted in substantially
higher levels of apoptotic cells, while treatment of parental
HEK293T cells did not, see Table 35. Neither the control ADC nor
huE22 mAb induced apoptosis to a significant degree. Thus,
huE22-AcBut-CM induced apoptosis in target cells in a target- and
calicheamicin-dependent manner.
TABLE-US-00035 TABLE 35 Apoptosis in target cells. HEK293T-EFNA4
HEK293T Parental Conc. % early % late % total % early % late %
total Compound (ng/mL) apoptosis apoptosis apoptosis apoptosis
apoptosis apoptosis None NA 4.9 1.7 6.6 1.6 0.7 2.3 (vehicle)
huE22- 1 22.4 7.8 30.2 6.0 1.6 7.6 AcBut-CM 100 19.0 10.2 29.2 4.0
1.6 5.6 1000 17.9 8.6 26.5 6.0 3.4 9.4 Control 1 5.5 4.0 9.5 3.3
1.4 4.7 ADC 100 4.5 2.1 6.6 3.7 0.9 4.6 1000 12.0 5.6 17.6 7.6 3.9
11.5 huE22 1 4.7 3.1 7.8 2.7 1.3 4.0 mAb 100 6.4 2.1 8.5 2.8 1.1
3.9 1000 8.3 2.3 10.6 1.8 0.8 2.6
Example 12
Enrichment of TNBC TPC
[0454] A PDX tumor bank containing 19 breast PDX tumors was
established. Based on patient pathology reports, tumor
histopathology, and microarray sub-clustering using the PAM-50(+)
panel, 13 of these PDX tumor models were confirmed to originate
from patients with TNBC; three of which were characterized to be of
the claudin-low (CL) subtype. Breast PDX tumors were harvested,
dissociated to single cell suspensions and analyzed by flow
cytometry using strict doublet discrimination gating. Further,
human ESA+ tumor cells were analyzed for CD46, CD324, CD24 and CD34
expression, respectively.
[0455] Upon careful phenotypic profiling for the critical cell
surface marker thought to demarcate tumor perpetuating cells (La
cancer stem cells) in breast cancer, CD24, it became apparent that
all cells in PDX tumors originating from patients with TNBC
uniformly express the antigen, and thus it held little utility for
identifying TPC in this subtype of breast cancer. Phenotypic
profiling of hundreds of cell surface antigens by flow cytometry
identified a number of heterogeneously expressed antigens,
including CD46 and CD324, in BR22 and BR31 PDX tumors.
[0456] Prospective TPC (i.e. ESA.sup.+CD46.sup.+CD324.sup.+ cells)
were isolated from BR22 PDX tumors by FACS and re-analyzed by flow
cytometry prior to implantation. Daughter tumor(s) were similarly
dissociated and analyzed to confirm cellular heterogeneity
reflecting the parental tumor. Tumor growth curves for individual
mice implanted with 50 ESA.sup.+CD46.sup.+CD324.sup.+
(diagonal-hatched circles) or ESA.sup.+CD46.sup.+CD324.sup.- (open
circles) cells isolated from dissociated BR22 and BR31 breast PDX
tumors, as shown in FIGS. 23 and 24, respectively. Utilization of
these markers to facilitate isolation and transplantation of single
cells without or with expression of CD46 and/or CD324 into
immunocompromised mice demonstrated that only
ESA.sup.+CD46.sup.+CD324.sup.+ cells, but not their CD324.sup.-
counterparts, were able to efficiently perpetuate tumors
replicating the phenotypic heterogeneity of their parental
tumors.
[0457] Fully heterogeneous tumors were efficiently initiated with
as few as 50 implanted cells, whereas TPC frequency within the
CD324.sup.- subpopulation was within error of the expected false
positive expectation resulting from a 1% cell impurity profile of
cells isolated on a BD FACSAria, see Table 36.
TABLE-US-00036 TABLE 36 ESA+ CD46+ PDX # Cells CD324+ CD324- BR13
50 10/20 50% 2/20 10% BR22 50 53/78 68% 2/41 5% BR31 50 13/30 43%
0/15 0% BR56 50 9/16 56% 2/13 15% BR86 100 11/20 55% 0/11 0% 200
8/12 67% 0/3 0% TG+ Mice 104 6 Mice Implanted 176 103 59% 6%
Functional TPC Frequency 1:71 cells 1:1,000 cells TPC Enrichment
Factor >14-fold
[0458] The demonstrated TPC frequency of 1:71 among the CD324.sup.+
cell subpopulation would yield an average 1.4 functional TPC per
mouse transplanted with 100 CD324.sup.+ cells, whereas a 1% sort
error rate would yield an average 0.014% CD324.sup.+ cell
contamination frequency among isolated CD324.sup.- cells, which
translates to a false positive frequency of 1.4% (0.2-2.7%
Range).
[0459] Further, whole transcriptome sequencing was done using the
Illumina HiSeq 2000 platform (100.times.100 bp paired-end
sequencing) using isolated ESA.sup.+CD46.sup.+CD324.sup.+ TPC and
ESA.sup.+ CD46.sup.+CD324.sup.- NTG cells, respectively, from a
number of breast PDX tumor models of various subtypes, including
claudin low and non-claudin low TNBC, and the Luminal B subtype of
breast cancer. Whole transcriptome sequencing was also performed
using mRNA obtained from normal tissues such as heart, liver,
kidney, lung, colon, skin, pancreas, and ovary. Resulting fpkm
(fragments per kilobase per million) values for all samples were
normalized using standard techniques and then filtered to focus on
genes encoding proteins annotated to be on the cell surface. Using
this filtered data set as input, the DESeq2 package within
Bioconductor was used to identify genes differentially expressed in
TPC versus both NTG cells and normal tissue, along with their
adjusted p-values. Notably, EFNA4 was significantly elevated in TPC
versus NTG cells and normal tissues, even after adjusting p-values
for false discovery rate.
Example 13
EFNA4 Expression is Elevated in TNBC TPC
[0460] To further the demonstrated enrichment of TNBC TPC, whole
transcriptome sequencing was performed with the Illumina HiSeq 2000
platform (100.times.100 bp paired-end sequencing) using isolated
ESA.sup.+CD46.sup.+CD324.sup.+ TPC and
ESA.sup.+CD46.sup.+CD324.sup.- NTG cells from a number of breast
PDX tumor models of various subtypes, including claudin low and
non-claudin low TNBC, and the Luminal B subtype of breast
cancer.
[0461] Whole transcriptome sequencing was also performed using mRNA
obtained from a panel of normal tissues such as heart, liver,
kidney, lung, colon, skin, pancreas, and ovary. Resulting FPKM
(fragments per kilobase per million) values for all samples were
normalized using standard techniques and then filtered to focus on
genes encoding proteins annotated to be on the cell surface. Using
this filtered data set as input, the DESeq2 package within
Bioconductor was used to identify genes differentially expressed in
TPC versus both NTG cells and normal tissue, along with their
adjusted p-values. Notably, EFNA4 was significantly elevated in TPC
versus NTG cells and normal tissues, even after adjusting p-values
for false discovery rate.
[0462] In addition, among the 19 breast PDX tumors encompassing
various subtypes (described in Example 12), EFNA4 mRNA expression
as assessed in bulk breast PDX tumors or normal vital organs by
microarray. EFNA4 mRNA expression was elevated an average of
greater than 2.6-fold higher than normal breast or any other normal
tissue assessed.
[0463] Further, expression of EFNA4 mRNA in normal-adjacent breast,
TNBC, and non-TNBC breast tumors were assed using available The
Cancer Genome Atlas (TCGA) Research Network (Weinstein JN et al.
Nature Genetics 45:1113-1120, 2013) data. EFNA4 expression was
generally higher in the non-Claudin low subtype of TNBC versus
other breast cancer subtypes. Upon applying the PAM(50)+ gene
signature to TOGA data generated from primary tumor specimens from
292 patients, elevated expression of EFNA4 in TNBC tumors (n=95)
remained evident compared to normal adjacent breast (n=108), and
breast tumors of the non-TNBC subtypes (n=197), as shown in FIG.
25. The horizontal lines indicate the median values.
[0464] The genetic basis of overexpression of EFNA4 in breast
cancer and ovarian cancer was analyzed. FIG. 26 shows the EFNA4
copy number in breast tumor samples and FIG. 27 shows the EFNA4
copy number in ovarian tumor samples from TOGA and METABRIC
(Curtis, C et al. Nature April. 18; 486(7403):346-352, 2012)
datasets. The white box represents the 25.sup.th-75.sup.th
percentiles, the error bars demarcate the 10.sup.th-90.sup.th
percentiles, and the individual dots fall below the 10.sup.th or
above the 90.sup.th percentile. The normal copy number (n=2) is
shown by the dashed line.
[0465] In some breast tumors the overexpression of EFNA4 may be a
consequence of copy number gain, see FIG. 26. In TCGA breast cancer
dataset 25.5% of tumor samples had notable copy number gain of
EFNA4 (n.gtoreq.2.5), while none had substantial copy number loss.
The subset of TNBC samples had a proportional incidence of EFNA4
copy number gain. The same trend was observed in the METABRIC
breast cancer dataset with n.gtoreq.2.5 in 14.3% of tumor samples.
Moreover, there is a strong correlation between EFNA4 mRNA level
and DNA copy number (r=0.48; p<0.0001). The data suggests a
potential genetic basis for the overexpression of EFNA4 in breast
cancer, and particularly TNBC.
[0466] To determine whether or not elevated EFNA4 gene expression
translated to an increase in protein, a panel of monoclonal
antibodies was generated by traditional immunization and hybridoma
approaches, and sandwich ELISA pairs were identified. Analysis of
tissue protein lysates from 17 normal organs, 49 primary breast
tumor specimens, and 9 TNBC PDX tumor models resulted in the
observation that EFNA4 protein levels were elevated, not only in
TNBC vs. normal tissues and other subtypes of breast cancer, but
expression was higher in the non-claudin-low subclass of TNBC
versus the claudin-low subset. These results confirm that, even at
the bulk tumor level, elevated EFNA4 gene expression translates to
meaningful increases in EFNA4 protein.
[0467] In ovarian cancer, the EFNA4 copy number gain (n.gtoreq.2.5)
was observed in a substantial fraction of TCGA samples (22.2%)
whereas there were no cases of significant copy number loss, see
FIG. 27. There is a strong correlation between EFNA4 mRNA level and
DNA copy number (r=0.32; p<0.0001), which was similar to the
observation in breast cancer. Together with the data in breast
cancer, this observation suggests a potential genetic basis for
EFNA4 overexpression in tumors.
[0468] In addition to breast and ovarian cancer, liver cancer
exhibited a high frequency of EFNA4 copy number gain. FIG. 28 shows
EFNA4 copy number in hepatocellular carcinoma (HCC) tumor samples
from 3 datasets: Pfizer-ACRG (n=310), Pfizer-Samsung (n=272) and
TCGA (n=212). Every sample is represented by a triangle, and the
horizontal lines indicate the median values. The normal copy number
(n=2) is shown by the dashed line. Consistently across three
datasets, there was frequent EFNA4 copy number gain but no
significant copy number loss; 68.4%, 37.5% and 29.7% of samples had
n.gtoreq.2.5 in the ACRG, Samsung and TCGA datasets respectively.
In liver cancer, there was a strong correlation between EFNA4 mRNA
level and DNA copy number in 3 independent datasets: TCGA
correlation coefficient=0.50 (p.about.0); ACRG coefficient=0.56
(p.about.0) and Samsung coefficient=0.38 (p=3E-09). EFNA4 gain in
HCC was not focal and instead occurred in the context of
amplification of the chromosomal region Chr1q21-1q22, which also
includes ELK4, MDM4 and PARP1. Notably, EFNA4 mRNA expression level
correlated strongly with copy number in the HCC datasets, which
suggested that tumors with EFNA4 gain may be more responsive to
treatment with an anti-EFNA4 ADC.
Example 14
Binding Properties of Ephrin-A4
[0469] The properties of Ephrin-A4 binding to Eph receptors were
further analyzed. Since anti-EFNA4 antibodies bind to Ephrin-A4
while the ligand is engaged with its Eph receptor, the affinity
between these two macromolecules was determined. The affinities of
commercially available Eph receptors and Ephrin-A4 were directly
compared by surface plasmon resonance using a BIAcore 2000 (GE
Healthcare) to determine the most relevant receptor. An anti-human
antibody capture kit was used to immobilize recombinant Eph
receptors on a CM5 biosensor chip. Prior to each antigen injection
cycle, Eph receptors at the concentration of 2 .mu.g/mL were
captured on the surface with a contact time of 2 minutes and a flow
rate of 5 .mu.L/min. The captured antibody loading from baseline
was constant at 150-260 response units. Following receptor capture
and 1 min baseline, monomeric human Ephrin-A4 was flowed over the
surface at concentrations of 400, 241, 145, 87, 53, 32, 19 and 12
nM for a 2 minute association phase followed by a 2 minute
dissociation phase at a flow rate of 10 .mu.L/min. Following each
cycle, the anti-human capture surface was regenerated with 30
seconds contact time of 3M MgCl.sub.2 at 10 .mu.L/min.
[0470] Biacore data was processed by initially calculating an
average of the response at equilibrium (Req). The Req was then
plotted versus concentration (M) and the steady state affinity fit
was used to calculate the affinity and theoretical Rmax. In all
cases, at least 5 different analyte concentrations were used to
make this calculation. All data analysis steps were completed in
BiaEvaluation Software 3.1 (GE Healthcare).
[0471] Raw data and Req versus concentration curves (not shown)
from the Ephrin-A4 binding to Eph receptors determined that the
receptor with the highest affinity was EphA2 and the receptor with
the lowest affinity was EphA10. Table 37 shows the nine Eph
receptors tested for binding to Ephrin-A4 in order of decreasing
affinity. The data demonstrates that the affinities for the Eph
receptors to Ephrin-A4 are all lower than the anti-EFNA4 antibodies
disclosed herein.
TABLE-US-00037 TABLE 37 Receptor Kd (nM) EphA2 31 EphA3 77 EphA6 82
EphA7 86 EphB3 108 EphA4 122 EphB2 135 EphA1 414 EphA10 431
Sequence CWU 1
1
13811276DNAHomo sapiens 1cttccctctt cactttgtac ctttctctcc
tcgactgtga agcgggccgg gacctgccag 60gccagaccaa accggacctc gggggcgatg
cggctgctgc ccctgctgcg gactgtcctc 120tgggccgcgt tcctcggctc
ccctctgcgc gggggctcca gcctccgcca cgtagtctac 180tggaactcca
gtaaccccag gttgcttcga ggagacgccg tggtggagct gggcctcaac
240gattacctag acattgtctg cccccactac gaaggcccag ggccccctga
gggccccgag 300acgtttgctt tgtacatggt ggactggcca ggctatgagt
cctgccaggc agagggcccc 360cgggcctaca agcgctgggt gtgctccctg
ccctttggcc atgttcaatt ctcagagaag 420attcagcgct tcacaccctt
ctccctcggc tttgagttct tacctggaga gacttactac 480tacatctcgg
tgcccactcc agagagttct ggccagtgct tgaggctcca ggtgtctgtc
540tgctgcaagg agaggaagtc tgagtcagcc catcctgttg ggagccctgg
agagagtggc 600acatcagggt ggcgaggggg ggacactccc agccccctct
gtctcttgct attactgctg 660cttctgattc ttcgtcttct gcgaattctg
tgagccaagc agaccttccc tctcatccca 720aggagccaga gtcctcccaa
gatcccctgg aggaggaggg atccctgctg cctgcactgg 780gggtgccaat
tcagaccgac aagatggagc attgatgggg gagatcagag ggtctgaggt
840gactcttgca ggagcctgtc ccctcatcac aggctaaaga agagcagtag
acagccctgg 900acactctgaa gcagaggcaa gacaaacaca ggcgctttgc
aggctgctct gagggtctca 960gcccatcccc caggaggact gggatttggt
atgatcaaat cctcaagcca gctgggggcc 1020caggctgaag acctggggac
aggtcgattg ctggaccagg gcaaagaaga agccctgcca 1080tctgtgccct
gtgggccttt tccctggggc agcaccttgc cctccccagg ggatcactca
1140cttgtcttct atgaagacgg actcttcatg aggttgaatt tcatgccagt
ttgtattttt 1200ataagtatct agaccaaacc ttcaataaac cactcatctt
tttgttgccc tccccaaaaa 1260aaaaaaaaaa aaaaaa 12762201PRTHomo sapiens
2Met Arg Leu Leu Pro Leu Leu Arg Thr Val Leu Trp Ala Ala Phe Leu 1
5 10 15 Gly Ser Pro Leu Arg Gly Gly Ser Ser Leu Arg His Val Val Tyr
Trp 20 25 30 Asn Ser Ser Asn Pro Arg Leu Leu Arg Gly Asp Ala Val
Val Glu Leu 35 40 45 Gly Leu Asn Asp Tyr Leu Asp Ile Val Cys Pro
His Tyr Glu Gly Pro 50 55 60 Gly Pro Pro Glu Gly Pro Glu Thr Phe
Ala Leu Tyr Met Val Asp Trp 65 70 75 80 Pro Gly Tyr Glu Ser Cys Gln
Ala Glu Gly Pro Arg Ala Tyr Lys Arg 85 90 95 Trp Val Cys Ser Leu
Pro Phe Gly His Val Gln Phe Ser Glu Lys Ile 100 105 110 Gln Arg Phe
Thr Pro Phe Ser Leu Gly Phe Glu Phe Leu Pro Gly Glu 115 120 125 Thr
Tyr Tyr Tyr Ile Ser Val Pro Thr Pro Glu Ser Ser Gly Gln Cys 130 135
140 Leu Arg Leu Gln Val Ser Val Cys Cys Lys Glu Arg Lys Ser Glu Ser
145 150 155 160 Ala His Pro Val Gly Ser Pro Gly Glu Ser Gly Thr Ser
Gly Trp Arg 165 170 175 Gly Gly Asp Thr Pro Ser Pro Leu Cys Leu Leu
Leu Leu Leu Leu Leu 180 185 190 Leu Ile Leu Arg Leu Leu Arg Ile Leu
195 200 3207PRTHomo sapiens 3Met Arg Leu Leu Pro Leu Leu Arg Thr
Val Leu Trp Ala Ala Phe Leu 1 5 10 15 Gly Ser Pro Leu Arg Gly Gly
Ser Ser Leu Arg His Val Val Tyr Trp 20 25 30 Asn Ser Ser Asn Pro
Arg Leu Leu Arg Gly Asp Ala Val Val Glu Leu 35 40 45 Gly Leu Asn
Asp Tyr Leu Asp Ile Val Cys Pro His Tyr Glu Gly Pro 50 55 60 Gly
Pro Pro Glu Gly Pro Glu Thr Phe Ala Leu Tyr Met Val Asp Trp 65 70
75 80 Pro Gly Tyr Glu Ser Cys Gln Ala Glu Gly Pro Arg Ala Tyr Lys
Arg 85 90 95 Trp Val Cys Ser Leu Pro Phe Gly His Val Gln Phe Ser
Glu Lys Ile 100 105 110 Gln Arg Phe Thr Pro Phe Ser Leu Gly Phe Glu
Phe Leu Pro Gly Glu 115 120 125 Thr Tyr Tyr Tyr Ile Ser Val Pro Thr
Pro Glu Ser Ser Gly Gln Cys 130 135 140 Leu Arg Leu Gln Val Ser Val
Cys Cys Lys Glu Arg Arg Ala Arg Val 145 150 155 160 Leu Pro Arg Ser
Pro Gly Gly Gly Gly Ile Pro Ala Ala Cys Thr Gly 165 170 175 Gly Ala
Asn Ser Asp Arg Gln Asp Gly Ala Leu Met Gly Glu Ile Arg 180 185 190
Gly Ser Glu Val Thr Leu Ala Gly Ala Cys Pro Leu Ile Thr Gly 195 200
205 4193PRTHomo sapiens 4Met Arg Leu Leu Pro Leu Leu Arg Thr Val
Leu Trp Ala Ala Phe Leu 1 5 10 15 Gly Ser Pro Leu Arg Gly Gly Ser
Ser Leu Arg His Val Val Tyr Trp 20 25 30 Asn Ser Ser Asn Pro Arg
Leu Leu Arg Gly Asp Ala Val Val Glu Leu 35 40 45 Gly Leu Asn Asp
Tyr Leu Asp Ile Val Cys Pro His Tyr Glu Gly Pro 50 55 60 Gly Pro
Pro Glu Gly Pro Glu Thr Phe Ala Leu Tyr Met Val Asp Trp 65 70 75 80
Pro Gly Tyr Glu Ser Cys Gln Ala Glu Gly Pro Arg Ala Tyr Lys Arg 85
90 95 Trp Val Cys Ser Leu Pro Phe Gly His Val Gln Phe Ser Glu Lys
Ile 100 105 110 Gln Arg Phe Thr Pro Phe Ser Leu Gly Phe Glu Phe Leu
Pro Gly Glu 115 120 125 Thr Tyr Tyr Tyr Ile Ser Val Pro Thr Pro Glu
Ser Ser Gly Gln Cys 130 135 140 Leu Arg Leu Gln Val Ser Val Cys Cys
Lys Glu Arg Asn Leu Pro Ser 145 150 155 160 His Pro Lys Glu Pro Glu
Ser Ser Gln Asp Pro Leu Glu Glu Glu Gly 165 170 175 Ser Leu Leu Pro
Ala Leu Gly Val Pro Ile Gln Thr Asp Lys Met Glu 180 185 190 His
5112PRTArtificial SequenceSynthetic peptide sequence 5Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Thr Thr Tyr 20 25
30 Gly Val Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Val Ile Trp Gly Gly Gly Ser Thr Asn Tyr Asn Ser Ala
Leu Lys 50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Ser Asp Trp Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 6335DNAArtificial
SequenceSynthetic nucleotide sequence 6gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccgtcact acttatggtg tggactgggt ccgccaagct 120ccagggaagg
ggctggagtg gttaggtgta atatggggtg gtggaagcac aaattataat
180agcgctttga agagccgatt caccatctcc agagacaact ccaagaacac
cctgtatctg 240caaatgaaca gtctgagagc cgaggacacg gccgtgtatt
actgtgccag tgattgggct 300tactggggcc aagggactct ggtcactgtc tcttc
3357107PRTArtificial SequenceSynthetic peptide sequence 7Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Gly Thr Asn 20
25 30 Val Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu
Ile 35 40 45 His Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Lys Arg Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 8322DNAArtificial SequenceSynthetic
nucleotide sequence 8gacatccaga tgacccagtc tccatcttcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gaatgtgggt acaaatgtag
cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatccattcg
gcatcctacc gttacagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttacta ttgtcagcaa tataagaggt atccgtacac gttcggaggg
300gggaccaagc tggaaataaa ac 3229122PRTArtificial SequenceSynthetic
peptide sequence 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly
Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe
Lys Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Thr Arg His Asp Pro Asn Asp Gly Tyr Tyr Phe Leu Phe Ala Tyr Trp 100
105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
10365DNAArtificial SequenceSynthetic nucleotide sequence
10gaggtgcaac tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt acctatggca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcgcaacc attagtagtg gtggtactta
cacatactac 180ccagactcag tgaagggccg attcaaaatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtac aagacatgac 300cccaatgatg gttactactt
cctgtttgct tactggggcc aggggactct ggtcactgtc 360tcttc
36511107PRTArtificial SequenceSynthetic peptide sequence 11Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Gly Asn Asn 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln His Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys
Leu Glu Ile Lys 100 105 12322DNAArtificial SequenceSynthetic
nucleotide sequence 12gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca aggccagtca gagtgttggc aacaatgtag
cttggtacca gcagaaacct 120ggccaggctc ccaggctcct catctactat
gcatccaata ggtatacagg catcccagac 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag actggagcct 240gaagattttg
cagtgtatta ctgtcaacag cattatagct ctccgctcac gttcggtgct
300gggaccaagc tggagatcaa ac 32213120PRTArtificial SequenceSynthetic
peptide sequence 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly
Asn Phe Asn Thr Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val
Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Glu Asp Gly Ser Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 14360DNAArtificial
SequenceSynthetic nucleotide sequence 14caggttcagc tggtgcagtc
tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta
cacctttacc ggctattaca tccactgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg atctaccctg gcaattttaa cacaaaatat
180aacgagcggt tcaagggcag agtcaccatg accacagaca catccacgag
cacagcctac 240atggagctga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagagaggat 300ggtagcccct actatgctat ggactactgg
ggtcaaggaa cctcagtcac cgtctcctca 360155PRTArtificial
SequenceSynthetic peptide sequence 15Gly Tyr Tyr Ile His 1 5
167PRTArtificial SequenceSynthetic peptide sequence 16Gly Tyr Thr
Phe Thr Gly Tyr 1 5 1715DNAArtificial SequenceSynthetic nucleotide
sequence 17ggctattaca tccac 151821DNAArtificial SequenceSynthetic
nucleotide sequence 18ggttacacct ttaccggcta t 211917PRTArtificial
SequenceSynthetic peptide sequence 19Trp Ile Tyr Pro Gly Asn Phe
Asn Thr Lys Tyr Asn Glu Arg Phe Lys 1 5 10 15 Gly 206PRTArtificial
SequenceSynthetic peptide sequence 20Tyr Pro Gly Asn Phe Asn 1 5
2151DNAArtificial SequenceSynthetic nucleotide sequence
21tggatctacc ctggcaattt taacacaaaa tataacgagc ggttcaaggg c
512218DNAArtificial SequenceSynthetic nucleotide sequence
22taccctggca attttaac 182311PRTArtificial SequenceSynthetic peptide
sequence 23Glu Asp Gly Ser Pro Tyr Tyr Ala Met Asp Tyr 1 5 10
2433DNAArtificial SequenceSynthetic nucleotide sequence
24gaggatggta gcccctacta tgctatggac tac 3325449PRTArtificial
SequenceSynthetic peptide sequence 25Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Tyr Pro Gly Asn Phe Asn Thr Lys Tyr Asn Glu Arg Phe 50 55
60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Asp Gly Ser Pro Tyr Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445 Gly 261347DNAArtificial
SequenceSynthetic nucleotide sequence 26caggttcagc tggtgcagtc
tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta
cacctttacc ggctattaca tccactgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg atctaccctg gcaattttaa cacaaaatat
180aacgagcggt tcaagggcag agtcaccatg accacagaca catccacgag
cacagcctac 240atggagctga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagagaggat 300ggtagcccct actatgctat ggactactgg
ggtcaaggaa cctcagtcac cgtctcctca 360gcctccacca agggcccatc
ggtcttcccc ctggcgccct cgagcaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgagccggt gacggtgtcg
480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 540ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag
1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc tccgggt 134727112PRTArtificial SequenceSynthetic peptide
sequence 27Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr
Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Phe Leu Tyr Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Thr His
Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
28336DNAArtificial SequenceSynthetic nucleotide sequence
28gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc
60atctcctgcc ggtctagtca gagcctcgtg catagtaatg gaaacacctt tttgtattgg
120tacctgcaga agccaggcca gtctccacag ctcctaatct atagagtttc
caaccggttc 180tctggagtgc cagataggtt cagtggcagc gggtcaggga
cagatttcac actgaaaatc 240agccgggtgg aggctgagga tgttggggtt
tattactgct ttcaagctac acatgttccg 300tggacgttcg gtggaggcac
caaagtggaa atcaaa 3362916PRTArtificial SequenceSynthetic peptide
sequence 29Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Phe
Leu Tyr 1 5 10 15 3011PRTArtificial SequenceSynthetic peptide
sequence 30Gln Ser Leu Val His Ser Asn Gly Asn Thr Phe 1 5 10
3148DNAArtificial SequenceSynthetic nucleotide sequence
31cggtctagtc agagcctcgt gcatagtaat ggaaacacct ttttgtat
483233DNAArtificial SequenceSynthetic nucleotide sequence
32cagagcctcg tgcatagtaa tggaaacacc ttt 33337PRTArtificial
SequenceSynthetic peptide sequence 33Arg Val Ser Asn Arg Phe Ser 1
5 3421DNAArtificial SequenceSynthetic nucleotide sequence
34agagtttcca accggttctc t 21359PRTArtificial SequenceSynthetic
peptide sequence 35Phe Gln Ala Thr His Val Pro Trp Thr 1 5
3624DNAArtificial SequenceSynthetic nucleotide sequence
36caagctacac atgttccgtg gacg 2437219PRTArtificial SequenceSynthetic
peptide sequence 37Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser
Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Phe Leu Tyr Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95
Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 38657DNAArtificial
SequenceSynthetic nucleotide sequence 38gatattgtga tgacccagac
tccactctct ctgtccgtca cccctggaca gccggcctcc 60atctcctgcc ggtctagtca
gagcctcgtg catagtaatg gaaacacctt tttgtattgg 120tacctgcaga
agccaggcca gtctccacag ctcctaatct atagagtttc caaccggttc
180tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac
actgaaaatc 240agccgggtgg aggctgagga tgttggggtt tattactgct
ttcaagctac acatgttccg 300tggacgttcg gtggaggcac caaagtggaa
atcaaacgga ctgtggctgc accaagtgtc 360ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact
tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa
480tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac
ctacagcctc 540agcagcaccc tgacgctgag caaagcagac tacgagaaac
acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc
acaaagagct tcaacagggg agagtgt 65739118PRTArtificial
SequenceSynthetic peptide sequence 39Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Tyr Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly
Gln Ile Asn Pro Asn Asn Gly Gly Thr Ala Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Trp Val Gly Thr His Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
40354DNAArtificial SequenceSynthetic nucleotide sequence
40caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt
60tcctgcaagg catctggata caccttcact tacttctata tgaactgggt gcgacaggcc
120cctggacaag ggcttgagtg ggtgggacaa atcaacccta ataatggtgg
cacagcctac 180gcacagaagt tccagggcag agtcaccatg accagggaca
cgtccacgag cacagtctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagatgggtc 300gggactcact actttgacta
ctggggccaa ggcaccactc tcacagtctc ctcc 354415PRTArtificial
SequenceSynthetic peptide sequence 41Tyr Phe Tyr Met Asn 1 5
427PRTArtificial SequenceSynthetic peptide sequence 42Gly Tyr Thr
Phe Thr Tyr Phe 1 5 4315DNAArtificial SequenceSynthetic nucleotide
sequence 43tacttctata tgaac 154421DNAArtificial SequenceSynthetic
nucleotide sequence 44ggatacacct tcacttactt c 214517PRTArtificial
SequenceSynthetic peptide sequence 45Gln Ile Asn Pro Asn Asn Gly
Gly Thr Ala Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 467PRTArtificial
SequenceSynthetic peptide sequence 46Asn Pro Asn Asn Gly Gly Thr 1
5 4751DNAArtificial SequenceSynthetic nucleotide sequence
47caaatcaacc ctaataatgg tggcacagcc tacgcacaga agttccaggg c
514821DNAArtificial SequenceSynthetic nucleotide sequence
48aaccctaata atggtggcac a 21499PRTArtificial SequenceSynthetic
peptide sequence 49Trp Val Gly Thr His Tyr Phe Asp Tyr 1 5
5027DNAArtificial SequenceSynthetic nucleotide sequence
50tgggtcggga ctcactactt tgactac 2751447PRTArtificial
SequenceSynthetic peptide sequence 51Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Tyr Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly
Gln Ile Asn Pro Asn Asn Gly Gly Thr Ala Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Trp Val Gly Thr His Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 521341DNAArtificial SequenceSynthetic nucleotide sequence
52caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt
60tcctgcaagg catctggata caccttcact tacttctata tgaactgggt gcgacaggcc
120cctggacaag ggcttgagtg ggtgggacaa atcaacccta ataatggtgg
cacagcctac 180gcacagaagt tccagggcag agtcaccatg accagggaca
cgtccacgag cacagtctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagatgggtc 300gggactcact actttgacta
ctggggccaa ggcaccactc tcacagtctc ctccgcctcc 360accaagggcc
catcggtctt ccccctggcg ccctcgagca agagcacctc tgggggcaca
420gcggccctgg gctgcctggt caaggactac ttccccgagc cggtgacggt
gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc 540tactccctca gcagcgtggt gaccgtgccc
tccagcagct tgggcaccca gacctacatc 600tgcaacgtga atcacaagcc
cagcaacacc aaggtggaca agaaagttga gcccaaatct 660tgtgacaaaa
ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca
720gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 900taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 960aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
1020aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga
tgagctgacc 1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 1140gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct
ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag
1320agcctctccc tgtctccggg t 134153111PRTArtificial
SequenceSynthetic peptide sequence 53Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Ser Tyr Thr
Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg
Leu Leu Ile Asn Phe Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His
Ser Trp 85 90 95 Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 54333DNAArtificial SequenceSynthetic
nucleotide sequence 54gaaattgtgt tgacacagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctctagct
atacttacat tcactggtac 120caacagaaac ctggccaggc tcccaggctc
ctcatcaatt ttgcatccaa cttggaaagt 180ggcatcccag ccaggttcag
tggcagtggg tctgggacag acttcactct caccatcagc 240agcctagagc
ctgaagattt tgcagtttat tactgtcagc acagttggga gattcctccg
300acgttcggtg gaggcaccaa gctggaaatc aaa 3335515PRTArtificial
SequenceSynthetic peptide sequence 55Arg Ala Ser Gln Ser Val Ser
Ser Ser Ser Tyr Thr Tyr Ile His 1 5 10 15 568PRTArtificial
SequenceSynthetic peptide sequence 56Ser Ser Ser Tyr Thr Tyr Ile
His 1 5 5745DNAArtificial SequenceSynthetic nucleotide sequence
57agggccagtc agagtgttag cagctctagc tatacttaca ttcac
455824DNAArtificial SequenceSynthetic nucleotide sequence
58agctctagct atacttacat tcac 24597PRTArtificial SequenceSynthetic
peptide sequence 59Phe Ala Ser Asn Leu Glu Ser 1 5
6021DNAArtificial SequenceSynthetic nucleotide sequence
60tttgcatcca acttggaaag t 21619PRTArtificial SequenceSynthetic
peptide sequence 61Gln His Ser Trp Glu Ile Pro Pro Thr 1 5
6227DNAArtificial SequenceSynthetic nucleotide sequence
62cagcacagtt gggagattcc tccgacg 2763218PRTArtificial
SequenceSynthetic peptide sequence 63Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Ser Tyr Thr
Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg
Leu Leu Ile Asn Phe Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His
Ser Trp 85 90 95 Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185
190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
64654DNAArtificial SequenceSynthetic nucleotide sequence
64gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agctctagct atacttacat tcactggtac
120caacagaaac ctggccaggc tcccaggctc ctcatcaatt ttgcatccaa
cttggaaagt 180ggcatcccag ccaggttcag tggcagtggg tctgggacag
acttcactct caccatcagc 240agcctagagc ctgaagattt tgcagtttat
tactgtcagc acagttggga gattcctccg 300acgttcggtg gaggcaccaa
gctggaaatc aaacggactg tggctgcacc aagtgtcttc 360atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg
420aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc
cctccaatcg 480ggtaactccc aggagagtgt cacagagcag gacagcaagg
acagcaccta cagcctcagc 540agcaccctga cgctgagcaa agcagactac
gagaaacaca aagtctacgc ctgcgaagtc 600acccatcagg gcctgagctc
gcccgtcaca aagagcttca acaggggaga gtgt 654658PRTMus spretus 65Gly
Tyr Thr Phe Thr Asp Tyr Glu 1 5 668PRTMus spretus 66Gly Phe Ser Leu
Thr Thr Tyr Gly 1 5 678PRTMus spretus 67Gly Tyr Thr Phe Thr Asn Tyr
Trp 1 5 688PRTMus spretus 68Gly Phe Thr Phe Ser Thr Tyr Gly 1 5
698PRTMus spretus 69Gly Tyr Thr Phe Thr Gly Tyr Tyr 1 5 708PRTMus
spretus 70Gly Tyr Thr Phe Thr Arg Asp Trp 1 5 718PRTMus spretus
71Gly Tyr Thr Phe Thr Tyr Phe Tyr 1 5 728PRTMus spretus 72Gly Tyr
Ser Phe Thr Val Tyr Asn 1 5 739PRTMus spretus 73Gly Tyr Ser Ile Thr
Ser Gly Tyr Tyr 1 5 748PRTMus spretus 74Gly Tyr Thr Phe Thr Gly Tyr
Tyr 1 5 758PRTMus spretus 75Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5
768PRTMus spretus 76Gly Ala Ser Ile Thr Ser Gly Tyr 1 5 778PRTMus
spretus 77Phe Asp Pro Glu Thr Gly Asn Thr 1 5 787PRTMus spretus
78Ile Trp Gly Gly Gly Ser Thr 1 5 798PRTMus spretus 79Ile Asp Pro
Ser Asp Ser Tyr Ile 1 5 808PRTMus spretus 80Ile Ser Ser Gly Gly Thr
Tyr Thr 1 5 818PRTMus spretus 81Ile Tyr Pro Gly Asn Phe Asn Thr 1 5
828PRTMus spretus 82Ile His Pro Tyr Asp Ser Glu Thr 1 5 838PRTMus
spretus 83Ile Asn Pro Asn Asn Gly Gly Thr 1 5 848PRTMus spretus
84Ile Asn Pro Tyr Tyr Gly Gly Thr 1 5 857PRTMus spretus 85Ile Ser
Tyr Asp Gly Arg Asn 1 5 868PRTMus spretus 86Ile Tyr Pro Gly Asn Phe
Asn Thr 1 5 878PRTMus spretus 87Ile His Pro Asn Ser Asp Thr Ile 1 5
887PRTMus spretus 88Ile Asn Tyr Ser Gly Asn Thr 1 5 8910PRTMus
spretus 89Ala Arg Gly Tyr Pro Ala Trp Phe Gly Tyr 1 5 10 906PRTMus
spretus 90Ala Ser Asp Trp Ala Tyr 1 5 9111PRTMus spretus 91Ala Arg
Glu Arg Leu Ser His Ala Met Asp Tyr 1 5 10 9215PRTMus spretus 92Thr
Arg His Asp Pro Asn Asp Gly Tyr Tyr Phe Leu Phe Ala Tyr 1 5 10 15
9313PRTMus spretus 93Ala Arg Glu Asp Gly Ser Pro Tyr Tyr Ala Met
Asp Tyr 1 5 10 9417PRTMus spretus 94Val Thr Phe Ile Lys Thr Met Val
Asp Thr Tyr Tyr Tyr Ala Met Asp 1 5 10 15 Tyr 9511PRTMus spretus
95Ala Arg Trp Val Gly Thr His Tyr Phe Asp Tyr 1 5 10 9615PRTMus
spretus 96Ala Arg Gly Gly Lys Thr Gly Thr Tyr Tyr Tyr Val Met Asp
Tyr 1 5 10 15 9712PRTMus spretus 97Ala Arg Glu Gly Tyr Gly Asp Tyr
Pro Phe Asp Tyr 1 5 10 9813PRTMus spretus 98Ala Arg Glu Asp Gly Ser
Pro Tyr Tyr Ala Met Asp Tyr 1 5 10 9911PRTMus spretus 99Ala Thr Pro
Glu Arg Arg Arg Ala Met Asp Tyr 1 5 10 10014PRTMus spretus 100Ala
Arg Ser Thr Met Ile Thr Thr Gly Ala Trp Phe Ala Tyr 1 5 10
10111PRTMus spretus 101Gln Ser Leu Ala His Thr Asn Gly Asn Thr Tyr
1 5 10 1026PRTMus spretus 102Gln Asn Val Gly Thr Asn 1 5 1036PRTMus
spretus 103Gln Asp Ile Lys Ser Tyr 1 5 1046PRTMus spretus 104Gln
Ser Val Gly Asn Asn 1 5 10511PRTMus spretus 105Gln Ser Leu Val His
Ser Asn Gly Asn Thr Phe 1 5 10 10611PRTMus spretus 106Gln Ser Leu
Leu His Ser Asp Gly Lys Thr Tyr 1 5 10 10710PRTMus spretus 107Gln
Ser Val Ser Ser Ser Ser Tyr Thr Tyr 1 5 10 1086PRTMus spretus
108Glu Asn Ile Asp Ser Tyr 1 5 10910PRTMus spretus 109Gln Ser Val
Ser Ser Ser Ser Tyr Ser Tyr 1 5 10 11011PRTMus spretus 110Gln Ser
Leu Val His Ser Asn Gly Asn Thr Phe 1 5 10 1117PRTMus spretus
111Ser Ser Leu Ser Ser Ser Tyr 1 5 1126PRTMus spretus 112Gln Ser
Val Ser Lys Asp 1 5 1138PRTMus spretus 113Lys Val Ser Asn Met Arg
Phe Ser 1 5 1147PRTMus spretus 114Ser Ala Ser Tyr Arg Tyr Ser 1 5
1157PRTMus spretus 115Tyr Ala Thr Ser Leu Ala Asp 1 5 1167PRTMus
spretus 116Tyr Ala Ser Asn Arg Tyr Thr 1 5 1177PRTMus spretus
117Arg Val Ser Asn Arg Phe Ser 1 5 1187PRTMus spretus 118Leu Val
Ser Asn Leu Asp Ser 1 5 1197PRTMus spretus 119Phe Ala Ser Asn Leu
Glu Ser 1 5 1207PRTMus spretus 120Ala Ala Thr Leu Leu Ala Asp 1 5
1217PRTMus spretus 121Tyr Ala Ser Asn Leu Glu Ser 1 5 1227PRTMus
spretus 122Arg Val Ser Asn Arg Phe Ser 1 5 1237PRTMus spretus
123Ser Thr Ser Phe Leu Ala Ser 1 5 1247PRTMus spretus 124Tyr Ala
Ser Asn Arg Tyr Thr 1 5 1259PRTMus spretus 125Ser Gln Asp Thr His
Val Pro Pro Thr 1 5 1269PRTMus spretus 126Gln Gln Tyr Lys Arg Tyr
Pro Tyr Thr 1 5 1279PRTMus spretus 127Leu Gln His Gly Glu Ser Pro
Tyr Thr 1 5 1289PRTMus spretus 128Gln Gln His Tyr Ser Ser Pro Leu
Thr 1 5 1299PRTMus spretus 129Phe Gln Ala Thr His Val Pro Trp Thr 1
5 1309PRTMus spretus 130Trp Gln Gly Thr His Phe Pro Gln Thr 1 5
1319PRTMus spretus 131Gln His Ser Trp Glu Ile Pro Pro Thr 1 5
1328PRTMus spretus 132Gln His Tyr Tyr Ser Thr Leu Thr 1 5
1339PRTMus spretus 133Gln His Ser Trp Glu Ile Pro Arg Thr 1 5
1349PRTMus spretus 134Phe Gln Ala Thr His Val Pro Trp Thr 1 5
1359PRTMus spretus 135Gln Gln Tyr Asp Ser Ser Pro Phe Thr 1 5
1369PRTMus spretus 136Gln Gln Asp Tyr Ser Ser Pro Leu Thr 1 5
13711PRTHomo sapiens 137Gln Arg Phe Thr Pro Phe Ser Leu Gly Phe Glu
1 5 10 13810PRTHomo sapiens 138Arg Leu Leu Arg Gly Asp Ala Val Val
Glu 1 5 10
* * * * *
References