U.S. patent application number 15/194926 was filed with the patent office on 2016-12-22 for polypeptide constructs and uses thereof.
The applicant listed for this patent is Teva Pharmaceuticals Australia Pty Ltd. Invention is credited to Adam Clarke, Anthony G. Doyle, Wouter Korver, Jack Tzu Chiao Lin, Glen E. Mikesell, Sarah L. Pogue, Matthew Pollard, Tetsuya Taura, Stephen Tran, David S. Wilson.
Application Number | 20160367695 15/194926 |
Document ID | / |
Family ID | 48168677 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160367695 |
Kind Code |
A1 |
Wilson; David S. ; et
al. |
December 22, 2016 |
POLYPEPTIDE CONSTRUCTS AND USES THEREOF
Abstract
The present invention provides a polypeptide construct
comprising a peptide or polypeptide signaling ligand linked to an
antibody or antigen binding portion thereof which binds to a cell
surface-associated antigen, wherein the ligand comprises at least
one amino acid substitution or deletion which reduces its potency
on cells lacking expression of said antigen.
Inventors: |
Wilson; David S.; (Freemont,
CA) ; Pogue; Sarah L.; (Freemont, CA) ;
Mikesell; Glen E.; (Pacifica, CA) ; Taura;
Tetsuya; (Palo Alto, CA) ; Korver; Wouter;
(Mountain View, CA) ; Doyle; Anthony G.;
(Drummoyne, AU) ; Clarke; Adam; (Five Dock,
AU) ; Pollard; Matthew; (Dural, AU) ; Tran;
Stephen; (Strathfield South, AU) ; Lin; Jack Tzu
Chiao; (Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Teva Pharmaceuticals Australia Pty Ltd |
Macquarie Park |
|
AU |
|
|
Family ID: |
48168677 |
Appl. No.: |
15/194926 |
Filed: |
June 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14262841 |
Apr 28, 2014 |
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15194926 |
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PCT/AU2012/001323 |
Oct 29, 2012 |
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14262841 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/55 20130101;
C07K 14/565 20130101; C07K 2317/33 20130101; C07K 2319/00 20130101;
C07K 2317/24 20130101; C07K 16/2896 20130101; C07K 14/5412
20130101; C07K 2319/30 20130101; A61P 35/00 20180101; C07K 14/5406
20130101; C07K 16/2887 20130101; C07K 2317/565 20130101; C07K
2319/33 20130101; C07K 14/57 20130101; C07K 16/28 20130101; C07K
2317/92 20130101; C07K 14/56 20130101; C07K 2319/21 20130101; A61K
47/642 20170801; C07K 2319/74 20130101; C07K 16/1027 20130101; C07K
16/46 20130101; A61K 38/00 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2011 |
AU |
2011904502 |
Claims
1. A polypeptide construct, comprising an antibody that
specifically binds to CD38, or antigen binding portion thereof,
linked to an attenuated interferon .alpha.2b (IFN .alpha.2b)
comprising at least one attenuating amino acid substitution.
2. The polypeptide construct according to claim 1, wherein the at
least one attenuating substitution is selected is selected from the
group consisting of R144A (SEQ ID NO: 30), R144S (SEQ ID NO: 40),
R144T (SEQ ID NO: 41), R144Y (SEQ ID NO: 43), R144I (SEQ ID NO:
35), R144L (SEQ ID NO: 37), A145D (SEQ ID NO: 44), A145G (SEQ ID
NO: 46), A145H (SEQ ID NO: 47), A145Y (SEQ ID NO: 58), A145K (SEQ
ID NO: 49), R33A, +YNS (SEQ ID NO: 65), R33A (SEQ ID NO: 16) and
R144A+YNS (SEQ ID NO: 68).
3. The polypeptide construct according to claim 1, wherein the
attenuated IFN .alpha.2b comprises the substitution A145D.
4. The polypeptide construct according to claim 3, wherein the
attenuated IFN .alpha.2b has the amino acid sequence of SEQ ID NO:
44.
5. The polypeptide construct according to claim 1, wherein the
antibody that specifically binds to CD38 comprises a heavy chain
comprising an HCDR1 having the amino acid sequence DSVMN, an HCDR2
having the amino acid sequence WIDPEYGRTDVAEKFKK, an HCDR3 having
the amino acid sequence TKYNSGYGFPY, an LCDR1 having the amino acid
sequence KASQNVDSDVD, an LCDR2 having the amino acid sequence
KASNRYT, and an LCDR3 having the amino acid sequence MQSNTHPRT.
6. The polypeptide construct according to claim 5, wherein the
attenuated IFN .alpha.2b comprises the substitution A145D.
7. The polypeptide construct according to claim 1, wherein the
antibody that specifically binds to CD38 comprises a variable light
chain and variable heavy chain pair selected from the group
consisting of SEQ ID NO: 384 and SEQ ID NO: 385, SEQ ID NO: 386 and
SEQ ID NO: 387, SEQ ID NO: 388 and SEQ ID NO: 389, SEQ ID NO: 390
and SEQ ID NO: 391, SEQ ID NO: 392 and SEQ ID NO: 393, SEQ ID NO:
394 and SEQ ID NO: 395, and SEQ ID NO: 396 and SEQ ID NO: 397.
8. The polypeptide according to claim 1, wherein the antibody that
specifically binds to CD38 comprises a variable light chain and
variable heavy chain pair selected from the group consisting of SEQ
ID NO: 134 and SEQ ID NO: 135, SEQ ID NO: 218 and SEQ ID NO: 219,
SEQ ID NO: 222 and SEQ ID NO: 223, SEQ ID NO: 226 and SEQ ID NO:
227, SEQ ID NO: 234 and SEQ ID NO: 235, SEQ ID NO: 242 and SEQ ID
NO: 243, and SEQ ID NO: 250 and SEQ ID NO: 251.
9. The polypeptide construct according to claim 7, wherein the
attenuated IFN .alpha.2b comprises the substitution A145D.
10. The polypeptide construct according to claim 8, wherein the
attenuated IFN .alpha.2b comprises the substitution A145D.
11. The polypeptide construct according to claim 7, wherein the
attenuated IFN .alpha.2b has the amino acid sequence of SEQ ID NO:
44.
12. The polypeptide construct according to claim 1, wherein the
attenuated IFN .alpha.2b is linked to the antibody or antigen
binding portion thereof via a peptide bond.
13. The polypeptide construct according to claim 1, wherein the
attenuated IFN .alpha.2b is linked to the antibody or antigen
binding portion thereof directly, or via a linker of 1 to 20 amino
acids in length.
14. The polypeptide construct according to claim 1, wherein the
antibody or antigen binding portion thereof comprises a light chain
and the attenuated IFN .alpha.2b is linked to the C-terminus of the
light chain.
15. The polypeptide construct according to claim 1, wherein the
antibody or antigen binding portion thereof comprises a heavy chain
and the attenuated IFN .alpha.2b is linked to the C-terminus of the
heavy chain.
16. The polypeptide construct according to claim 1, wherein the
antibody or antigen binding portion thereof specifically binds to
CD38 with an affinity of from 50 nM to 1 pM, from 25 nM to 1 pM,
from 10 nM to 1 pM, or from 5 nM to 1 pM.
17. The polypeptide construct according to claim 1, wherein the
antigen binding portion of the antibody comprises an Fab.
18. The polypeptide construct according to claim 1, wherein the
construct has an Antigen-Specificity Index of greater than 50.
19. The polypeptide construct according to claim 1, wherein the
antibody comprises a human IgG1 heavy chain constant domain.
20. The polypeptide construct according to claim 1, wherein the
antibody comprises a human IgG4 heavy chain constant domain.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/262,841, filed on Apr. 28, 2014, which is a
continuation of International Patent Application No.
PCT/AU2012/001323, filed on Oct. 29, 2012, which claims priority to
Australian Patent Application No. 2011904502, filed on Oct. 28,
2011, the contents of each application are incorporated herein by
reference in their entirety and for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
electronically as a text file named Pctau2012001323-seql.txt,
created on Apr. 24, 2014, with a size of 1 megabyte. The Sequence
Listing is incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to polypeptide constructs
comprising mutated, attenuated polypeptide ligands attached to
antibodies, wherein the antibodies direct the mutated ligands to
cells that express on their surfaces the antigens to which said
antibodies bind, as well as receptors for said ligands. The
invention further relates to methods of treatment involving the use
of these polypeptide constructs.
BACKGROUND OF THE INVENTION
[0004] Numerous peptide and polypeptide ligands have been described
to function by interacting with a receptor on a cell surface, and
thereby stimulating, inhibiting, or otherwise modulating a
biological response, usually involving signal transduction pathways
inside the cell that bears the said receptor. Examples of such
ligands include peptide and polypeptide hormones, cytokines,
chemokines, growth factors, apoptosis-inducing factors and the
like. Natural ligands can be either soluble or can be attached to
the surface of another cell.
[0005] Due to the biological activity of such ligands, some have
potential use as therapeutics. Several peptide or polypeptide
ligands have been approved by regulatory agencies as therapeutic
products, including, for example, human growth hormone, insulin,
interferon (IFN)-.alpha.2b, IFN.alpha.2.alpha., IFN.beta.,
erythropoietin, G-CSF and GM-CSF. Many of these and other ligands
have demonstrated potential in therapeutic applications, but have
also exhibited toxicity when administered to human patients. One
reason for toxicity is that most of these ligands trigger receptors
on a variety of cells, including cells other than those that
mediate the therapeutic effect. For example, when IFN.alpha.2b is
used to treat multiple myeloma its utility resides, at least in
part, in its binding to type I interferon receptors on the myeloma
cells, which in turn triggers reduced proliferation and hence
limits disease progression. Unfortunately, however, this IFN also
binds to numerous other, normal cells within the body, triggering a
variety of other cellular responses, some of which are harmful
(e.g. flu-like symptoms, neutropenia, depression). A consequence of
such "off target" activity of ligands is that many ligands are not
suitable as drug candidates. In this context, "off target activity"
refers to activity on the ligand's natural receptor, but on the
surface of cells other than those that mediate therapeutically
beneficial effects.
[0006] Even though some ligands, such as IFN.alpha.2b, are approved
for the treatment of medical conditions, they are poorly tolerated
due to their "off target" biological activity. The off-target
activity and associated poor tolerability also mean that some of
these peptide ligand-based drugs cannot be administered at
sufficiently high dosages to produce optimal therapeutic effects on
the target cells which mediate the therapeutic effect.
[0007] Similarly, it has been known since the mid-1980's that
interferons, in particular IFN.alpha., are able to increase
apoptosis and decrease proliferation of certain cancer cells. These
biological activities are mediated by type I interferon receptors
on the surface of the cancer cells which, when stimulated, initiate
various signal transduction pathways leading to reduced
proliferation and/or the induction of terminal differentiation or
apoptosis. IFN.alpha. has been approved by the FDA for the
treatment of several cancers including melanoma, renal cell
carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenous
leukemia (CML) and hairy cell leukemia. A "direct" effect of
IFN.alpha. on the tumour cells is mediated by the IFN.alpha.
binding directly to the type I IFN receptor on those cells and
stimulating apoptosis, terminal differentiation or reduced
proliferation. One "indirect" effect of IFN.alpha. on non-cancer
cells is to stimulate the immune system, which may produce an
additional anti-cancer effect by causing the immune system to
reject the tumour.
[0008] Unfortunately, the type I interferon receptor is also
present on most non-cancerous cells. Activation of this receptor on
such cells by IFN.alpha. causes the expression of numerous
pro-inflammatory cytokines and chemokines, leading to toxicity.
Such toxicity prevents the dosing of IFN.alpha. to a subject at
levels that exert the maximum anti-proliferative and pro-apoptotic
activity on the cancer cells.
[0009] Ozzello et al. (Breast Cancer Research and Treatment
25:265-76, 1993) described covalently attaching human IFN.alpha. to
a tumour-targeting antibody, thereby localizing the direct
inhibitory activity of IFN.alpha. to the tumour as a way of
reducing tumour growth rates, and demonstrated that such conjugates
have anti-tumour activity in a xenograft model of a human cancer.
The mechanism of the observed anti-cancer activity was attributed
to a direct effect of IFN.alpha. on the cancer cells, since the
human IFN.alpha. used in the experiments did not interact
appreciably with the murine type I IFN receptor, which could have
lead to an indirect anti-cancer effect. Because of this lack of
binding of the human IFN.alpha. to the murine cells, however, the
authors could not evaluate the toxicity of the antibody-IFN.alpha.
conjugate relative to free INF.alpha.. These authors used a
chemical method to attach the IFN.alpha. to the antibody.
[0010] Alkan et al., (Journal of Interferon Research, volume 4,
number 3, p. 355-63, 1984) demonstrated that attaching human
IFN.alpha. to an antibody that binds to the Epstein-Barr virus
(EBV) membrane antigen (MA) increased its antiproliferative
activities towards cells that express the EBV-MA antigen. This
increased potency was dependent on both antigen expression by the
target cells and the binding specificity of the antibody. The cell
line tested was the cancer cell line QIMR-WIL, a myeloblastic
leukemia. The authors suggested that the attachment of IFN.alpha.
to an antibody could be used as a treatment for cancer since it
would reduce tumour growth. Alkan et at did not address the
potential toxicity of these antibody-IFN.alpha. conjugates arising
from their interactions with normal, antigen-negative cells.
[0011] It is also known that the linkage between an antibody and
IFN.alpha. may be accomplished by making a fusion protein
construct. For example, IDEC (WO01/97844) disclose a direct fusion
of human IFN.alpha. to the C terminus of the heavy chain of an IgG
targeting the tumour antigen CD20. Other groups have disclosed the
use of various linkers between the C-terminus of an IgG heavy chain
and the IFN.alpha.. For example, U.S. Pat. No. 7,456,257 discloses
that the C-terminus of an antibody heavy chain constant region may
be connected to IFN.alpha. via an intervening serine-glycine rich
(S/G) linker of the sequence (GGGGS).sub.n, where n may be 1, 2 or
3, and that there are no significant differences in the IFN.alpha.
activity of the fusion protein construct regardless of linker
length.
[0012] Morrison et al. (US2011/0104112 A1; and Xuan C, Steward K K,
Timmerman J M, Morrison S L. Targeted delivery of
interferon-.alpha. via fusion to anti-CD20 results in potent
antitumor activity against B-cell lymphoma. Blood 2010;
115:2864-71) also disclose IFN.alpha. linked to the C-terminus of
the heavy chain of a cancer-targeting IgG antibody, with an
intervening S/G linker, and observed that the fusion of the IgG and
linker to the IFN.alpha. reduced the activity of IFN.alpha. on
cells that did not express the corresponding antigen on the cell
surface. The decreased IFN activity of these fusion protein
constructs was modest when compared to human non-fusion protein
IFN.alpha. (free IFN.alpha.) acting on human cells, but appeared to
be more significant for murine IFN.alpha. on murine cells. The
decrease in the activity of human IFN.alpha. that results from
fusing it to the C-terminus of an antibody, as observed by Morrison
et al, and in U.S. Pat. No. 7,456,257 is modest and is generally
considered to be a disadvantage since it reduces potency of the
ligand. This disadvantage was pointed out, for example, by Rossi et
at (Blood vol. 114, No. 18, pp 3864-71), who used an alternative
strategy of attaching the IFN.alpha. to a tumor targeting antibody
in such a way that no loss in IFN.alpha. activity was observed.
[0013] In general the prior art teaches to use a potent IFN and to
target this IFN to cancer cells. While this approach results in an
increase in activity of the IFN against cancer cells, it does not
address the issue of activity of the IFN on normal "off-target"
cells. In prior art examples referred to above, the human
IFN.alpha. portion of the antibody-IFN.alpha. fusion protein
maintained a high proportion of native IFN.alpha. activity when
exposed to human cells that do not express the corresponding
antigen on their cell surfaces. This activity may lead to toxicity
arising from the activation of non-cancerous, normal ("off target")
cells by the IFN.alpha. portion of the fusion protein. Accordingly,
there exists a need to decrease the "off-target" activity of
ligand-based drugs, while retaining the "on-target", therapeutic
effect of such ligands. The maintenance of target-specific ligand
activity and at the same time a reduction in non-target toxicity of
ligand-based therapeutic agents would create a greater therapeutic
concentration window for therapeutically useful ligands. It would
for example be desirable to use human IFN.alpha. in a form such
that its activity can be directed to the cancer cells while
minimizing its effects on normal human cells. Ideally the type I
interferon receptor on the cancer cells would be maximally
stimulated, while the same receptor on non-cancerous cells would
experience minimal stimulation. There is a need to target human
IFN.alpha. to the cancer cells in such a way that it has
dramatically more activity on the cancer cells, which display the
antigen, than on the normal cells, which do not display the
antigen. The same logic applies to other potentially therapeutic
ligands, e.g. other cytokines, peptide and polypeptide hormones,
chemokines, growth factors, apoptosis-inducing factors and the
like.
SUMMARY OF THE INVENTION
[0014] The present inventors have found that when a peptide or
polypeptide signaling ligand, having one or more mutations which
substantially decrease the affinity of the ligand for its receptor,
is linked to an antibody that targets the mutated ligand to target
cells which display the antibody's corresponding antigen, the
ligand's activity on target antigen-positive cells is maintained
while the ligand's activity on non-target antigen-negative cells is
substantially reduced. The net result is a ligand signaling
molecule that has a much greater potency in activation of its
receptors on antigen-positive target cells compared to
antigen-negative non-target cells, which provides a means for
reducing toxicity arising from off-target ligand activity.
[0015] Accordingly, a first aspect of the present invention
provides a polypeptide construct comprising a peptide or
polypeptide signaling ligand linked to an antibody or antigen
binding portion thereof which binds to a cell surface-associated
antigen, wherein the ligand comprises at least one amino acid
substitution or deletion which reduces its potency on cells lacking
expression of said antigen.
[0016] In a second aspect, the present invention provides a method
of treating a tumour in a subject, comprising administering to the
subject the polypeptide construct of the present invention.
[0017] In a third aspect, the present invention provides use of the
polypeptide construct of the present invention in the treatment of
cancer.
[0018] In a fourth aspect, the present invention provides a
composition comprising the polypeptide construct of the present
invention and a pharmaceutically acceptable carrier or diluent.
[0019] In a fifth aspect, the present invention provides method of
reducing the potency of a peptide or polypeptide signaling ligand
on an antigen negative cell which bears the ligand receptor whilst
maintaining the potency of the ligand on an antigen positive cell
which bears the ligand receptor to a greater extent when compared
to the antigen negative cell, the method comprising modifying the
ligand such that the ligand comprises at least one amino acid
substitution or deletion which reduces its potency on the antigen
negative cell and linking the modified ligand to an antibody or
antigen-binding portion thereof, wherein the antibody or antigen
binding portion thereof is specific for a cell surface-associated
antigen on the antigen positive cell but not on the antigen
negative cell.
[0020] Unlike the linking of a non-attenuated "native" or
"wild-type" human ligand to an antibody or antigen-binding portion
thereof, which typically results in from 1 to 15-fold higher
potency of the ligand on antigen-positive compared to
antigen-negative cells, the present invention demonstrates that the
attachment of mutated, attenuated forms of the ligand to the same
antibody is able to generate higher potency on antigen-positive
cells compared to antigen negative cells.
[0021] In one embodiment the signaling ligand is IFN.alpha. or
IFN.beta. and the polypeptide construct shows at least 10, at least
100, at least 1,000, at least 10,000 or at least 100,000-fold
greater selectivity towards antigen positive cells over antigen
negative cells compared to free, wild-type ligand using the
"off-target" assay and the "on target (ARP)" or "on target (Daudi)"
assays described herein.
[0022] The present invention also provides an antibody-attenuated
ligand fusion proteins, wherein the attenuated ligand is IFN.alpha.
or IFN.beta. and the wherein fusion protein construct, when
injected into a mouse with an established human tumor, can
eliminate the tumor.
[0023] The present invention also provides an antibody-attenuated
ligand fusion proteins, wherein the attenuated ligand is IFN.alpha.
or IFN.beta. and wherein the fusion protein construct, when
injected into a mouse with an established human tumor with a volume
of over 500 cubic millimeters, can eliminate the tumor.
[0024] The present invention also provides an antibody-attenuated
ligand fusion proteins, wherein the attenuated ligand is IFN.alpha.
or IFN.beta. and wherein the fusion protein construct, when
injected as a single one-time treatment into a mouse with an
established human tumor, can eliminate the tumor.
[0025] An antibody-attenuated ligand fusion proteins, wherein the
attenuated ligand is IFN.alpha. or IFN.beta. and wherein the fusion
protein construct can eliminate both established myeloma tumors and
established lymphoma tumors in a mouse
[0026] In each of these cases it is preferred that cell
surface-associated antigen is CD 38.
[0027] In one embodiment, the amino acid sequence of the signaling
ligand comprising at least one amino acid substitution or deletion
has greater than 90% or greater than 95%, or greater than 96%, or
greater than 97%, or greater than 98% or greater than 99% sequence
identity with the wild-type ligand amino acid sequence.
[0028] In one embodiment, the construct is a fusion protein.
[0029] In certain embodiments the signaling ligand is linked to the
C-terminus of the heavy chain of the antibody or antigen binding
portion thereof. In certain embodiments the signaling ligand is
linked to the C-terminus of the light chain of the antibody or
antigen binding portion thereof. In either of these embodiments,
the ligand may be linked directly to the C-terminus of the heavy or
light chain of the antibody or antigen binding portion thereof (ie
without an intervening additional linker).
[0030] In one embodiment the cell surface associated antigen is
selected from class I MHC or PD-1.
[0031] In certain embodiments, the cell surface-associated antigen
is a myeloma associated antigen which is selected from the group
consisting of CD38, HM1.24, CD56, CS1, CD138, CD74, IL-6R, Blys
(BAFF), BCMA, HLA-SR, Kininogen, beta2 microglobulin, FGFR3,
ICAM-1, matriptase, CD52, EGFR, GM2, alpha4-integrin, IFG-1R and
KIR, and the ligand is an IFN.alpha..
[0032] In one embodiment, the signaling ligand is selected from any
one of IFN.alpha.2b, IFN.beta., IL-4 or IL-6.
[0033] In certain embodiments in which the signaling ligand is an
IFN.alpha., the amino acid substitution or deletion may be at any
one or more of amino acid positions R33, R144 or A145. In certain
embodiments the signaling ligand is an IFN.alpha. and the
substitution is selected from the group consisting of R144A (SEQ ID
NO:30), R144S (SEQ ID NO:40), R144T (SEQ ID NO:41), R144Y (SEQ ID
NO:43), R144I (SEQ ID NO:35), R144L (SEQ ID NO:37), A145D (SEQ ID
NO:44), A145H (SEQ ID NO:47), A145Y (SEQ ID NO:58), A145K (SEQ ID
NO:49), R33A+YNS (SEQ ID NO:65), R33A (SEQ ID NO:16) and R144A+YNS
(SEQ ID NO:68).
[0034] In certain embodiments in which the signaling ligand is an
IFN.alpha. and the cell surface associated antigen is CD38, the
antibody is selected from any one of G003, G005, G024, MOR03077,
MOR03079, MOR03080, MORO3100, 385B13, 385B18, 385B19, 385B30,
38SB31, 38SB39, OKT10, X355/02, X910/12, X355/07, X913/15, R5D1,
R5E8, R10A2, or an antigen binding portion thereof, or an antibody
with greater than 95%, greater than 96%, greater than 97%, greater
than 98% or at least 99% amino acid sequence identity with any one
of R5D1, R5E8 or R10A2.
[0035] In certain embodiments in which the cell surface associated
antigen is CD38, the signaling ligand of the polypeptide construct
is an IFN.alpha., the treatment is for a cancer in a subject
selected from multiple myeloma, a leukemia or a lymphoma. In
particular embodiments the subject is also treated with a retinoid,
such as all-trans retinoic acid. In certain embodiments in which
the cell surface associated antigen is CD38, the tumour or cancer
may be selected from multiple myeloma, non-Hodgkin's lymphoma,
chronic myelogenous leukemia, chronic lymphocytic leukemia or acute
myelogenous leukemia.
[0036] In embodiments in which the ligand is linked to an antibody,
the antibody may be an IgG4. In particular embodiments the IgG4
comprises an S228P amino acid substitution.
[0037] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IFN.alpha., the antibody or antigen
binding portion thereof may bind to a cell surface associated
antigen on virally infected cells. In these embodiments the cell
surface associated antigen may be selected from a virally encoded
protein, phosphatidylserine or a phosphatidylserine-binding
protein. In embodiments in which the cell surface associated
antigen is phosphatidylserine or a phosphatidylserine-binding
protein the construct may be used to treat Hepatitis C.
[0038] In certain embodiments in which the signaling ligand of the
polypeptide construct is IFN.alpha. or IFN.beta., the cell surface
associated antigen is selected from CD20, CD38, CD138 or CS1. In
certain embodiments in which the ligand is IFN.alpha. or IFN.beta.,
the tumour or cancer may be selected from multiple myeloma,
melanoma, renal cell carcinoma, chronic myelogenous leukemia or
hairy cell leukemia.
[0039] In a particular embodiment the construct is
G005-HC-L0-IFN.alpha. (A145D) IgG4.
[0040] In certain embodiments in which the signaling ligand of the
polypeptide constrct is an IFN.beta., the cell surface associated
antigen may be a T cell, a myeloid cell or an antigen presenting
cell surface associated protein.
[0041] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IFN.beta., the cell surface associated
antigen may be selected from the group consisting of CD3, CD4, CD8,
CD24, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS,
CD33, CD115, CD11c, CD14, CD52 and PD-1. In these embodiments, the
construct may be used to treat a disease characterized by excess
inflammation, such as an autoimmune disease.
[0042] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IFN.beta., the at least one amino acid
substitution or deletion is selected from the group consisting of
R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, R152H. In these
embodiments, the IFN.beta. may also possess a C17S or C17A
substitution.
[0043] In certain embodiments the signaling ligand of the
polypeptide construct is an IFN.gamma.. In these embodiments, the
cell surface associated antigen may be a tumor-associated antigen.
In other embodiments, the cell surface associated antigen may be
selected from the group consisting of CD14, FSP1, FAP, PDGFR alpha
and PDGFR beta. In these embodiments, the construct may be used to
treat a disease characterized by excess fibrosis.
[0044] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IFN.gamma., the at least one amino acid
substitution or deletion is selected from the group consisting of a
deletion of residues A23 and D24, an S20I substitution, an A23V
substitution, a D21K substitution and a D24A substitution.
[0045] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IL-4, the cell surface associated
antigen is selected from the group consisting of CD3, CD4, CD24,
CD38, CD44, CD69, CD71, CD96, PD-1, ICOS, CD52 and PD-1.
[0046] In certain embodiments in which the signaling ligand of the
polypeptide construct is an IL-6, the cell surface associated
antigen is selected from the group consisting of CD3, CD4, CD24,
CD38, CD44, CD69, CD71, CD96, PD-1, ICOS, CD52 and PD-1.
[0047] In certain embodiments in which the signaling ligand of the
polypeptide construct is an HGF, the cell surface associated
antigen is selected from the group consisting of ASGR1, ASGR2,
FSP1, RTI140/Ti-alpha, HTI56 and a VEGF receptor.
[0048] In certain embodiments in which the signaling ligand of the
polypeptide construct is a TGF.beta., the cell surface associated
antigen is selected from the group consisting of CD3, CD4, CD8,
CD24, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS,
CD33, CD115, CD11c, CD14, CD52 and PD-1.
[0049] In certain embodiments in which the signaling ligand of the
polypeptide construct is an erythropoietin, the cell surface
associated antigen is selected from the group consisting of CD241
the product of the RCHE gene, CD117 (c-kit), CD71 (transferrin
receptor), CD36 (thrombospondin receptor), CD34, CD45RO, CD45RA,
CD115, CD168, CD235, CD236, CD237, CD238, CD239 and CD240.
[0050] In certain embodiments in which the signaling ligand of the
polypeptide construct is an interleukin-10 and the cell surface
associated antigen is selected from the group consisting of CD11c,
CD33 or CD115, CD14, FSP1, FAP, or PDGFR (alpha or beta).
[0051] In a sixth aspect there is provided anti-CD38 antibodies
with variable regions designated X910/12, X913/15, X355/02,
X355/07, R5D1, R5E8, or R10A2, with sequences set out as
follows:
TABLE-US-00001 Name V.sub.H sequence V.sub.K/V.sub.L sequence
X910/12 SEQ ID NO: 395 SEQ ID NO: 394 X913/15 SEQ ID NO: 397 SEQ ID
NO: 396 X355/01 SEQ ID NO: 421 SEQ ID NO: 420 X355/02 SEQ ID NO:
391 SEQ ID NO: 390 X355/04 SEQ ID NO: 423 SEQ ID NO: 422 X355/07
SEQ ID NO: 393 SEQ ID NO: 392 R5D1 SEQ ID NO: 399 SEQ ID NO: 398
R5E8 SEQ ID NO: 401 SEQ ID NO: 400 R10A2 SEQ ID NO: 403 SEQ ID NO:
402
[0052] From these sequences the person skilled in the field can
readily identify the CDR sequences using known methods. As will be
recognized by the skilled worker these CDR sequences can be used in
differing framework sequences to those specified in the SEQ ID NO's
specified above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 shows a schematic of the certain embodiments of the
present invention that comprise an antibody consisting of 2 heavy
chains and 2 light chain, in which one or two attenuated signaling
ligands is or are attached to each heavy chain or each light chain,
or both.
[0054] FIG. 2 shows a schematic illustrating one possible approach
for how the antibody-attenuated ligand fusion proteins of the
present invention cause signaling by activating receptors on cells
that display the antigen corresponding to the said antibody on
their cell surfaces. The fusion protein activates the receptor on
the same cell that the antibody is bound to, via its specific
antigen.
[0055] FIG. 3 shows the amino acid sequences of the human CD38 (SEQ
ID NO:131).
[0056] FIGS. 4a-4b show the amino acid sequences of certain
exemplary signaling ligands of the present invention: (4a) human
IFN.alpha.2b, IFN.beta.1, IFN.beta.1b and IFN.gamma.; and (4b) IL-4
and IL-6.
[0057] FIGS. 5a-5e show the amino acid sequences of certain
antibody-attenuated ligand fusion proteins of the present
invention: (5a) G005-HC-L0-IFN.alpha. (A145D) IgG4; (5b)
nBT062-HC-L0-IFN.alpha. (A145D) IgG4; (5c) G005-HC-L0-IFN.beta.
(R35A) IgG4; (5d) HB95-HC-L16-IL-6 (R179E) IgG1; and (5e)
J110-HC-L6-IL-4 (R88Q) IgG1. The nomenclature for the fusion
proteins is described in the examples.
[0058] FIG. 6 shows the non-antibody-antigen-targeted interferon
activity of IFN.alpha.2b, and of the antibody-IFN fusion protein
constructs Rituximab-IFN.alpha.2b (Rituximab-HC-L6-IFN.alpha. IgG1)
and Palivizumab-IFN.alpha.2b (Isotype-HC-L6-IFN.alpha. IgG1) in the
interferon activity assay described in the examples below as the
"off-target assay. Throughout the figures "IFN.alpha. equivalents"
refers to the molar concentration of interferon molecules, either
free or attached to an antibody. "IFN" refers to free (non-fusion
protein) wild-type interferon.
[0059] FIG. 7 shows the antibody-antigen-targeted interferon
activity of the Rituximab-IFN.alpha.2b fusion protein construct
(Rituximab-HC-L6-IFN.alpha. IgG1) compared with IFN.alpha.2b in the
antiproliferative assay described in the examples below as the "on
target (Daudi) assay."
[0060] FIG. 8 shows the antibody-antigen-targeted interferon
activity of the Rituximab-IFN.alpha. fusion protein construct
(Rituximab-HC-L6-IFN.alpha. IgG1) compared with the non-targeted
activity of Palivizumab-IFN.alpha. fusion protein construct
(Isotype-HC-L6-IFN.alpha. IgG1) in the "on-target (Daudi) assay"
described in the examples below.
[0061] FIG. 9 shows the non-antibody-antigen-targeted interferon
activity of IFN.alpha.2b, of the antibody-IFN fusion protein
constructs Rituximab-IFN.alpha.2b (Rituximab-HC-L6-IFN.alpha. IgG1)
and Palivizumab-IFN.alpha.2b (Isotype-HC-L6-IFN.alpha. IgG1), and
of certain variants of Rituximab-IFN.alpha.2b constructs that have
been mutated to reduce their interferon activity. The assay is
described in the examples as the "off-target assay".
[0062] FIG. 10 shows the non-antibody-antigen-targeted interferon
activity of the antibody-IFN fusion protein constructs
Rituximab-IFN.alpha.2b (Rituximab-HC-L6-IFN.alpha. IgG1) and of two
variants of Rituximab-IFN.alpha.2b that were mutated to reduce
interferon activity. The assay is described in the examples as the
"off-target assay".
[0063] FIG. 11 shows the antibody-antigen-targeted interferon
activity of the antibody-IFN fusion protein construct
Rituximab-IFN.alpha.2b (Rituximab-HC-L6-IFN.alpha. IgG1) and of
variants of Rituximab-IFN.alpha.2b constructs that have been
mutated to reduce their interferon activity compared to the
non-targeted activity of the Palivizumab-IFN.alpha.2b
(Isotype-HC-L6-IFN.alpha. IgG1) fusion protein constructs and
compared to IFN.alpha.2b. The assay is described in the examples as
the "on target (Daudi) assay."
[0064] FIG. 12 shows the antibody-antigen-targeted interferon
activity of the antibody-IFN fusion protein constructs
Rituximab-IFN.alpha.2b (Rituximab-HC-L6-IFN.alpha. IgG1) and of two
variants that were mutated to reduce interferon activity. The assay
is described in the examples as the "on target (Daudi) assay."
[0065] FIG. 13 shows the sequences of certain novel human CD38
antibodies disclosed herein.
[0066] FIG. 14 shows the results of detection of binding of novel
human anti-CD38 antibodies to a CD38.sup.+ cell line RPMI-8226 by
flow cytometry. The x axis is the antibody concentration in
micrograms/ml and the y axis represents the mean fluorescence
intensity.
[0067] FIG. 15 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b compared with an anti-CD38-IFN.alpha. fusion
protein construct (G005-HC-L0-IFN.alpha. IgG4), based on the
anti-CD38 antibody G005. The assay is described in the examples as
the "off-target assay."
[0068] FIG. 16 shows the antiproliferative activity of IFN.alpha.2b
vs an anti-CD38-IFN.alpha. fusion protein construct
(G005-HC-L0-IFN.alpha. IgG4) on the multiple myeloma cell line
ARP-1 (CD38.sup.+). The assay is described in the examples as the
"on target (ARP) assay."
[0069] FIG. 17 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b vs various anti-CD38-IFN.alpha. fusion protein
constructs bearing point mutations in the IFN portion. The antibody
variable regions of these fusion protein constructs were derived
from antibody G005. The assay is described in the examples as the
"off-target assay."
[0070] FIG. 18 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b vs various anti-CD38-IFN.alpha. fusion protein
constructs bearing point mutations in the IFN portion. The antibody
variable regions of these fusion proteins were derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0071] FIG. 19 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b vs two anti-CD38-IFN.alpha. fusion protein
constructs bearing point mutations in the IFN portion. The antibody
variable regions of these fusion protein constructs are derived
from antibody G005. The assay is described in the examples as the
"off-target assay."
[0072] FIG. 20 shows the antiproliferative activity of IFN.alpha.2b
vs anti-CD38-IFN.alpha. fusion protein constructs with mutations in
the IFN portion on the lymphoma cell line Daudi. The antibody
variable regions of these fusion protein constructs are derived
from antibody G005. The assay is described in the examples as the
"on target (Daudi) assay."
[0073] FIG. 21 shows the anti-proliferative activity of
IFN.alpha.2b and various anti-CD38-IFN.alpha. fusion protein with
the A145G mutation in the IFN portion. Fusion protein constructs
have either the 6 amino acid L6 linker or no linker (L0) and are of
the IgG1 or IgG4 isotype. The antibody variable regions of these
fusion protein constructs are derived from antibody G005. The assay
is described in the examples as the "on target (Daudi) assay".
[0074] FIG. 22 shows the anti-proliferative activity of
IFN.alpha.2b and two anti-CD38-IFN.alpha. fusion protein with the
A145G mutation in the IFN portion. Both fusion protein constructs
had the IFN portion linked to the C-terminus of the light chain,
with either a six amino acid linker (L6) or no linker (L0). The
antibody variable regions of these fusion protein constructs are
derived from antibody G005. The assay is described in the examples
as the "on target (Daudi) assay."
[0075] FIG. 23 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the R144A mutation in the IFN
portion. The experiment compares the potency of the fusion protein
constructs as a function of isotype (IgG1 vs. IgG4) and the
presence or absence of the L6 linker between the antibody heavy
chain C-terminus and the N-terminus of the mutated IFN. The
antibody variable regions of these fusion protein constructs are
derived from antibody G005. The assay is described in the examples
as the "on target (ARP) assay."
[0076] FIG. 24 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the A145G mutation in the IFN
portion. The experiment compares the potency of the fusion protein
constructs as a function of isotype (IgG1 vs. IgG4) and the
presence or absence of the L6 linker between the antibody heavy
chain C-terminus and the N-terminus of the mutated IFN. The
antibody variable regions of these fusion protein constructs are
derived from antibody G005. The assay is described in the examples
as the "on target (ARP) assay."
[0077] FIG. 25 shows the non-antibody-antigen targeted IFN activity
of various anti-CD38-IFN.alpha. fusion protein constructs with
different point mutations in the IFN portion. The antibody variable
regions of these fusion protein constructs are derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0078] FIG. 26 shows the non-antibody-antigen targeted IFN activity
of various anti-CD38-IFN.alpha. fusion protein constructs with
different point mutations in the IFN portion. The antibody variable
regions of these fusion protein constructs are derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0079] FIG. 27 shows the non-antibody-antigen targeted IFN activity
of various anti-CD38-IFN.alpha. fusion protein constructs with
different point mutations in the IFN portion. The antibody variable
regions of these fusion protein constructs are derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0080] FIG. 28 shows the non-antibody-antigen targeted IFN activity
of various anti-CD38-IFN.alpha. fusion protein constructs with
different point mutations in the IFN portion. The antibody variable
regions of these fusion protein constructs are derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0081] FIG. 29 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b vs various anti-CD38-IFN.alpha. fusion protein
constructs with different point mutations in the IFN portion. The
antibody variable regions of these fusion protein constructs are
derived from antibody G005. The assay is described in the examples
as the "off-target assay."
[0082] FIG. 30 shows the non-antibody-antigen targeted IFN activity
of various anti-CD38-IFN.alpha. fusion protein constructs with
different point mutations in the IFN portion. The antibody variable
regions of these fusion protein constructs are derived from
antibody G005. The assay is described in the examples as the
"off-target assay."
[0083] FIG. 31 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of anti-CD38-IFN.alpha. fusion protein
constructs with the various mutations in the IFN portion. The
antibody variable regions of these fusion protein constructs are
derived from antibody G005. The assay is described in the examples
as the "on target (ARP) assay."
[0084] FIG. 32 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the various mutations in the IFN
portion. The antibody variable regions of these fusion protein
constructs are derived from antibody G005. The assay is described
in the examples as the "on target (ARP) assay.".
[0085] FIG. 33 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the R144A mutation in the IFN
portion. The experiment compares different antibody variable
regions in the context of the same mutated IFN fusion protein. The
assay is described in the examples as the "on target (ARP)
assay."
[0086] FIG. 34 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the A145D mutation in the IFN
portion. The experiment compares different antibody variable
regions in the context of the same mutated IFN fusion protein
construct. The assay is described in the examples as the "on target
(ARP) assay."
[0087] FIG. 35 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b and various anti-CD38-IFN.alpha. fusion protein
constructs with the R144A mutation in the IFN portion. The
experiment compares different antibody variable regions in the
context of the same mutated IFN fusion protein construct. The assay
is described in the examples as the "off-target assay."
[0088] FIG. 36 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b and various anti-CD38-IFN.alpha. fusion protein
constructs with the A145D mutation in the IFN portion. The
experiment compares different antibody variable regions in the
context of the same mutated IFN fusion protein construct. The assay
is described in the examples as the "off-target assay."
[0089] FIG. 37 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.alpha.2b vs anti-CD38-IFN.alpha.
fusion protein constructs with the A145D mutation in the IFN
portion. The experiment compares different antibody variable
regions in the context of the same mutated IFN fusion protein
construct. The assay is described in the examples as the "on target
(ARP) assay."
[0090] FIG. 38 shows the non-antibody-antigen targeted IFN activity
of IFN.alpha.2b and various anti-CD38-IFN.alpha. fusion protein
constructs with the A145D mutation in the IFN portion. The
experiment compares different antibody variable regions in the
context of the same mutated IFN fusion protein construct. The assay
is described in the examples as the "off-target assay."
[0091] FIG. 39 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of two antibody-IFN.alpha. fusion protein
constructs with the A145D mutation in the IFN portion. The nBT062
antibody binds CD138 whereas the "isotype" antibody does not (it is
derived from the antibody 2D12). The assay is described in the
examples as the "on target (ARP) assay."
[0092] FIGS. 40a-40b show the antiproliferative activity on the
multiple myeloma cell line ARP-1 of IFN.alpha.2b and two
antibody-IFN.alpha. fusion protein constructs with the A145D
mutation in the IFN portion. The HB95 antibody binds human class I
MHC (which is expressed on the ARP-1 cells) whereas the "isotype"
antibody does not (it is derived from the antibody 2D12).
Palivizumab, like 2D12, does not bind to the ARP-1 cells. FIG. 40b
shows the same assay, comparing antibody-attenuated IFN.alpha.
fusion protein constructs in which the antibody portion is a Fab
fragment rather than a full size antibody. For both FIGS. 40a and
40b, the assay is described in the examples as the "on target (ARP)
assay."
[0093] FIG. 41 shows measurements of the antiviral activity of
IFN.alpha. and two antibody-IFN.alpha. fusion protein constructs
with the A145D mutation in the IFN portion. This cytopathic effect
inhibition assay utilized the cell line A549 and the EMC virus. The
HB95 antibody binds human class I MHC (which is expressed on the
A549 cells) whereas the "isotype" antibody derived from the
antibody 2D12 does not.
[0094] FIG. 42 shows the non-antibody-antigen targeted IFN activity
of IFN.beta., an anti-CD38-IFN.beta. fusion protein construct and
an identical fusion protein construct but with the attenuating R35A
mutation in the IFN portion. The assay is described in the examples
as the "off-target assay."
[0095] FIG. 43 shows the antiproliferative activity on the multiple
myeloma cell line ARP-1 of IFN.beta., an anti-CD38-IFN.beta. fusion
protein construct and an identical fusion protein construct but
with the attenuating R35A mutation in the IFN portion. The antibody
variable regions of these fusion protein constructs are derived
from the antibody G005. The assay is described in the examples as
the "on target (ARP) assay." "Ifn equivalents" refers to the molar
concentration of interferon molecules, either free or attached to
an antibody.
[0096] FIG. 44 shows the non-antibody-antigen-targeted IL-4
activity ["off-target (HB-IL4) assay"] of IL-4 and three
antibody-IL-4 fusion protein constructs: J110-HC-L6-IL-4 IgG1, an
anti-PD1 antibody fused to wild type IL-4; J110-HC-L6-IL-4 (R88Q),
which is identical to the previously mentioned fusion protein
construct except for the attenuating R88Q mutation in the IL-4
portion; and Isotype-HC-L6-IL-4 (R88Q), based on the 2D12 antibody,
which does not bind to any of the cells used in the assays of the
present invention, and is fused to the attenuated IL-4. "IL-4
equivalents" refers to the molar concentration of IL-4 molecules,
either free or attached to an antibody.
[0097] FIG. 45 shows the "on target (Th1 diversion) assay"
comparing the activity of IL-4 and three antibody-IL-4 fusion
protein constructs: J110-HC-L6-IL-4 IgG1, an anti-PD1 antibody
fused to wild type IL-4; J110-HC-L6-IL-4 (R88Q), which is identical
to the previously mentioned fusion protein construct except for the
attenuating R88Q mutation in the IL-4 portion; and
Isotype-HC-L6-IL-4 (R88Q), based on the 2D12 antibody, which does
not bind to any of the cells used in the assays of the present
invention, and is fused to the attenuated IL-4. "IL-4 equivalents"
refers to the molar concentration of IL-4 molecules, either free or
attached to an antibody.
[0098] FIG. 46 shows the "IL-6 bioassay" comparing IL-6 with
various antibody-IL-6 fusion protein constructs that either do bind
to the target cells (based on the HB95 antibody, which binds to
class I MHC on the target cells) or do not bind the target cells
(based on the isotype control antibody 2D12), fused to either wild
type IL-6 or IL-6 with the attenuating R179E mutation. "IL-6
equivalents" refers to the molar concentration of IL-6 molecules,
either free or attached to an antibody.
[0099] FIG. 47 shows the effects of various compounds on the growth
of subcutaneous H929 myeloma tumors in SCID mice. The bar labeled
"treatment" shows the duration of treatment with the compounds. The
"isotype" antibody was based on antibody 2D12. G005 is an anti-CD38
antibody.
[0100] FIG. 48 shows the effects of various compounds on survival
(Kaplan-Meier graph) of NOD-SCID mice systemically inoculated with
the human myeloma cell line MM1S. The bar labeled "treatment" shows
the duration of treatment with the compounds. G005 is an anti-CD38
antibody.
[0101] FIG. 49 shows the effects of various compounds on the growth
of subcutaneous Daudi lymphoma tumors in NOD-SCID mice. The bar
labeled "treatment" shows the duration of treatment with the
compounds. The "isotype" antibody was based on antibody 2D12. G005
is an anti-CD38 antibody.
[0102] FIG. 50 shows the effects of an anti-CD38-attenuated
IFN.alpha. fusion protein construct (G005-HC-L6-IFN.alpha. (A145G)
IgG1) and an isotype control-attenuated IFN.alpha. fusion protein
construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG1) on the growth of
subcutaneous H929 myeloma tumors in SCID mice, at various doses.
The bar labeled "treatment" shows the duration of treatment with
the compounds. The "isotype" antibody was based on antibody
2D12.
[0103] FIG. 51 shows the effects of anti-CD38-attenuated IFN.alpha.
fusion protein constructs vs isotype control antibody-attenuated
IFN.alpha. fusion protein constructs, on the growth of subcutaneous
H929 myeloma tumors in SCID mice. IgG1 is compared to IgG4 in the
context of these fusion protein constructs. The bar labeled
"treatment" shows the duration of treatment with the compounds. The
"isotype" antibody was based on antibody 2D12.
[0104] FIG. 52 shows the effects of an anti-CD38-attenuated
IFN.alpha. fusion protein construct (X355/02-HC-L0-IFN.alpha.
(A145D) IgG4) vs an isotype control antibody-attenuated IFN.alpha.
fusion protein constructs on the growth of subcutaneous H929
myeloma tumors in SCID mice. The bar labeled "treatment phase"
shows the duration of treatment with the compounds. The "isotype"
antibody was based on antibody 2D12.
[0105] FIG. 53 shows the effects of various compounds on the growth
of subcutaneous H929 myeloma tumors in SCID mice. G005 is an
anti-CD38 antibody.
[0106] FIG. 54 shows the effects of an anti-CD38-attenuated
IFN.alpha. fusion protein construct (G005-HC-L6-IFN.alpha. (A145G)
IgG4) and an isotype control-attenuated IFN.alpha. fusion protein
construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG4) on the growth of
subcutaneous H929 myeloma tumors in SCID mice, with several rounds
of administration each at a dose of 10 mg/kg. The "isotype"
antibody was based on antibody 2D12.
[0107] FIG. 55 shows the effects of an anti-CD38-attenuated
IFN.alpha. fusion protein construct (G005-HC-L6-IFN.alpha. (A145G)
IgG4) on the growth of subcutaneous H929 myeloma tumors in SCID
mice. Dosing (indicated by arrows) was initiated when the median
tumor volume reached 730 mm.sup.3.
[0108] FIG. 56 shows the inhibition of colony formation from normal
human bone marrow mononuclear cells (BM MNC) by IFN.alpha.2b, an
anti-CD38-attenuated IFN.alpha. fusion protein construct
(G005-HC-L0-IFN.alpha. (A145D) IgG4) and an isotype control
antibody-attenuated IFN.alpha. fusion protein construct
2D12-HC-L0-IFN.alpha. (A145D) IgG4. The antibody-attenuated
IFN.alpha. fusion protein constructs show about 10,000-fold reduced
potency in this assay.
[0109] FIGS. 57a and 57b show the effects of IFN.alpha.2b vs an
antibody-attenuated IFN.alpha. fusion protein construct
(Isotype-HC-L6-IFN.alpha. (A145G) IgG1; the isotype variable
regions are based on antibody 2D12) on cytokine production by human
peripheral blood mononuclear cells (PBMCs). (57a) IP-10 and MCP-1;
(57b) MCP-3 and IL-1.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0110] The constructs of the present invention are
antibody-attenuated ligand constructs, which show an elevated
antigen-specificity index with respect to activating signaling
pathways due to the action of the attenuated ligand on a cell
surface receptor. These constructs are based on the surprising
discovery that, in the context of an antibody-ligand construct, the
ligand portion can be mutated in such a way that the ligand
activity on antigen-negative cells is dramatically attenuated,
while the ligand activity on antigen-positive cells is only
modestly, if at all, attenuated. Such constructs display one, two,
three, four or five orders of magnitude greater potency on
antigen-positive cells compared to antigen negative cells than does
the free ligand. In one embodiment, the antibody-attenuated ligand
construct retains at least 1%, at least 10%, at least 20%, at least
30%, at least 40% or at least 50% of the potency on
antigen-positive cells as the non-attenuated free (i.e. not
attached to an antibody) ligand. In addition, in one embodiment the
antibody-attenuated ligand construct retains at least 30%, at least
50%, at least 75% or at least 90% of the maximal activity of the
non-attenuated free (i.e. not attached to an antibody) ligand; in
this context, "maximal activity" should be understood as meaning
the amount of signaling activity (or downstream effect thereof) at
the high, plateau portion of a dose-response curve, where further
increases in the agent does not further increase the amount of
response).
[0111] "Specificity" as used herein is not necessarily an absolute
designation but often a relative term signifying the degree of
selectivity of an antibody-ligand fusion protein construct for an
antigen-positive cell compared to an antigen-negative cell. Thus
for example, a construct may be said to have "100-fold specificity
for antigen-positive cells compared to antigen-negative cells" and
this would indicate that the construct has 100-fold higher potency
on cells that express the antigen compared to cells that do not. In
some cases, this degree of specificity of a construct comparing
antigen-positive vs. antigen-negative cells may not be based on the
absolute ratio of potency of the construct on antigen-positive vs.
antigen-negative cells, but of the potency of the construct on each
type of cell relative to the potency of the free, non attenuated
ligand on the same type of cell. This "ratio of ratio" approach for
quantifying the degree of specificity of an antibody-ligand
construct takes into consideration any inherent differences in the
potency of a ligand on different cell types and is exemplified by
the calculations of Antigen Specificity Index (ASI) in Table 25.
Assays for determining potency of antibody-ligand fusion constructs
are exemplified in the examples and include cell based assays for
proliferation, apoptosis, phosphorylation of receptors and
intracellular proteins, migration, differentiation (for example,
differentiation of naive CD4+ T cells into Th1, Th17, Th2 vs. Treg
cells), increases or decreases in gene expression or gene product
secretion into the media, etc.
[0112] Accordingly, in a first aspect the present invention
provides a polypeptide construct comprising a peptide or
polypeptide signaling ligand linked to an antibody or antigen
binding portion thereof which binds to a cell surface-associated
antigen wherein the ligand comprises at least one amino acid
substitution or deletion which reduces its potency on cells lacking
expression of said antigen.
[0113] In one embodiment the present invention provides a
polypeptide construct comprising IFN linked to an antibody or
antigen binding portion thereof which binds to a tumour associated
antigen wherein the IFN comprises at least one amino acid
substitution or deletion which reduces its potency on cells lacking
expression of said antigen. Such a polypeptide will be capable of
exerting with high potency the IFN's anti-proliferative activity on
the antigen-positive tumor cells while exerting a much lower
potency on the antigen-negative, non-tumour cells within the
body.
[0114] In a second aspect the present invention provides a method
of treating a tumour in a subject comprising administering to the
subject the polypeptide construct of the present invention.
[0115] The term "antibody-ligand construct" as used herein refers
to an antibody or antigen-binding fragment thereof covalently
attached to a signaling ligand that has been attenuated by one or
more substitutions or deletions that reduce the ligand's potency on
cells that do not express the antigen corresponding to the
antibody.
[0116] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art,
non-limiting embodiments of which are discussed below.
[0117] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[0118] The term "antigen binding domain" or "antigen binding
portion" of an antibody, as used herein, refers to one or more
fragments of an antibody or protein that retain the ability to
specifically bind to an antigen (e.g., CD38). It has been shown
that the antigen-binding function of an antibody can be performed
by fragments of a full-length antibody. Such antibody embodiments
may also be bispecific, dual specific, or multi-specific formats,
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CHl domains; (ii) a
F(ab')2 fragment, a bivalent fragment comprising two Fab fragments
in addition to a portion of the hinge region, linked by a disulfide
bridge at the hinge region; (iii) an Fd fragment consisting of the
VH and CHl domains; (iv) an Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, (v) a domain antibody (dAb)
(Ward et al. 1989 Nature 341 544-6, Winter et al., PCT publication
WO 90/05144 Al herein incorporated by reference), which comprises a
single variable domain; and (vi) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, VL and VH, are coded for by separate genes, they
can be joined, using recombinant methods, by a synthetic linker
that enables them to be made as a single protein chain in which the
VL and VH regions pair to form monovalent molecules (known as
single chain Fv (scFv); see e.g., Bird et al. 1988 Science 242
423-6; Huston et al. 1988 Proc Natl Acad Sci USA 85 5879-83). Such
single chain antibodies are also intended to be encompassed within
the term "antigen-binding portion" of an antibody. Other forms of
single chain antibodies, such as diabodies, are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al., 1993, Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al., 1994, Structure
2:1121-1123). Such antibody binding portions are known in the art
(Kontermann and Dubel eds., Antibody Engineering 2001
Springer-Verlag. New York. 790 pp., ISBN 3-540-41354-5). In an
embodiment the antibody binding portion is a Fab fragment.
[0119] The antibody described herein may be may be a humanized
antibody. The term "humanized antibody" shall be understood to
refer to a protein comprising a human-like variable region, which
includes CDRs from an antibody from a non-human species (e.g.,
mouse or rat or non-human primate) grafted onto or inserted into
FRs from a human antibody (this type of antibody is also referred
to a "CDR-grafted antibody"). Humanized antibodies also include
proteins in which one or more residues of the human protein are
modified by one or more amino acid substitutions and/or one or more
FR residues of the human protein are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found in neither the human antibody or in the non-human
antibody. Any additional regions of the protein (e.g., Fc region)
are generally human. Humanization can be performed using a method
known in the art, e.g., U.S. Pat. No. 5,225,539, U.S. Pat. No.
6,054,297, U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585,089. The
term "humanized antibody" also encompasses a super-humanized
antibody, e.g., as described in U.S. Pat. No. 7,732,578.
[0120] The antibody described herein may be human. The term "human
antibody" as used herein refers to proteins having variable and,
optionally, constant antibody regions found in humans, e.g. in the
human germline or somatic cells or from libraries produced using
such regions. The "human" antibodies can include amino acid
residues not encoded by human sequences, e.g. mutations introduced
by random or site directed mutations in vitro (in particular
mutations which involve conservative substitutions or mutations in
a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5
of the residues of the protein). These "human antibodies" do not
necessarily need to be generated as a result of an immune response
of a human, rather, they can be generated using recombinant means
(e.g., screening a phage display library) and/or by a transgenic
animal (e.g., a mouse) comprising nucleic acid encoding human
antibody constant and/or variable regions and/or using guided
selection (e.g., as described in or U.S. Pat. No. 5,565,332). This
term also encompasses affinity matured forms of such antibodies.
For the purposes of the present disclosure, a human protein will
also be considered to include a protein comprising FRs from a human
antibody or FRs comprising sequences from a consensus sequence of
human FRs and in which one or more of the CDRs are random or
semi-random, e.g., as described in U.S. Pat. No. 6,300,064 and/or
U.S. Pat. No. 6,248,516.
[0121] The antibody portions of polypeptides of the present
invention may be full length antibodies of any class, preferably
IgG1, IgG2 or IgG4. The constant domains of such antibodies are
preferably human. The variable regions of such antibodies may be of
non-human origin or, preferably, be of human origin or be
humanized. Antibody fragments may also be used in place of the full
length antibodies.
[0122] The term "antibody" also includes engineered antibodies. As
will be appreciated there are many variations of engineered
antibodies (e.g. mouse monoclonal, chimeric, humanized and human
monoclonal antibodies, single chain variable antibody fragments
(scFv's), minibodies, aptamers, as well as bispecific antibodies
and diabodies as described above).
[0123] Single variable region domains (termed dAbs) are, for
example, disclosed in (Ward et al., Nature 341: 544-546, 1989;
Hamers-Casterman et al., Nature 363: 446-448, 1993; Davies &
Riechmann, FEBS Lett. 339: 285-290, 1994).
[0124] Minibodies are small versions of whole antibodies, which
encode in a single chain the essential elements of a whole
antibody. Suitably, the minibody is comprised of the VH and VL
domains of a native antibody fused to the hinge region and CH3
domain of the immunoglobulin molecule as, for example, disclosed in
U.S. Pat. No. 5,837,821.
[0125] In an alternate embodiment, the engineered antibody may
comprise non-immunoglobulin derived, protein frameworks. For
example, reference may be made to (Ku & Schutz, Proc. Natl.
Acad. Sci. USA 92: 6552-6556, 1995) which discloses a four-helix
bundle protein cytochrome b562 having two loops randomized to
create CDRs, which have been selected for antigen binding.
[0126] There is a plethora of non-antibody recognition protein or
protein domain scaffolds that may be utilised as the antigen
binding domains in the constructs of this invention. These include
scaffolds based on cytotoxic T lymphocyte-associated antigen 4
(CTLA-4) (Evibody; U.S. Pat. No. 7,166,697); human transferrin
(Trans-body); a three-helix bundle from the Z-domain of Protein A
(Affibody); a monomeric or trimeric human C-type lectin domain
(Tetranectin); the tenth human fibronectin type III domain
(AdNectin); the Kunitz-type domain of human or bovine trypsin
inhibitor; insect Defensin A (IICA29), APPI (Kuntiz domains);
lipocalins, FABP, Bilin-binding protein, Apoloproptein D
(Anticalins); human .alpha.-crystallin or ubiquitin molecule
(Affilin); trypsin inhibitor II (Microbody); .alpha.2p8 or Ankyrin
repeat (repeat-motif proteins), Charybdotoxin (Scorpion toxins),
Min-23, Cellulose binding domain (Knottins); Neocarzinostatin,
CBM4-2 and Tendamistat.
[0127] Further, in addition to scaffolds provided for by
antibody-derived domains or non-antibody folds as described above,
there are naturally occurring ligand binding proteins or protein
domains that may be utilised as the ligand binding domains in this
invention. For example, protein domains that possess ligand binding
properties include extracellular domains of receptors, PDZ modules
of signalling proteins, such as Ras-binding protein AF-6, adhesion
molecules, and enzymes.
[0128] The present invention further encompasses chemical analogues
of amino acids in the subject antibodies. The use of chemical
analogues of amino acids is useful inter alia to stabilize the
molecules such as if required to be administered to a subject. The
analogues of the amino acids contemplated herein include, but are
not limited to, modifications of side chains, incorporation of
unnatural amino acids and/or their derivatives during peptide,
polypeptide or protein synthesis and the use of crosslinkers and
other methods which impose conformational constraints on the
proteinaceous molecule or their analogues.
[0129] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with 2, 4,
6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups
with succinic anhydride and tetrahydrophthalic anhydride; and
pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0130] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0131] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivatisation, for example, to a corresponding amide.
[0132] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0133] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0134] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0135] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acid,
contemplated herein is shown in Table 1.
TABLE-US-00002 TABLE 1 Non-conventional Non-conventional amino acid
Code amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-Nmethylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcylcopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm
N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
[0136] Crosslinkers can be used, for example, to stabilize 3D
conformations, using homo-bifunctional crosslinkers such as the
bifunctional imido esters having (CH2)n spacer groups with n=1 to
n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an
amino-reactive moiety such as N-hydroxysuccinimide and another
group specific-reactive moiety such as maleimido or dithio moiety
(SH) or carbodiimide (COOH).
[0137] Using methods well known in the art to increase binding, by
for example, affinity maturation, or to decrease immunogenicity by
removing predicted MHC class II-binding motifs. The therapeutic
utility of the antibodies described herein can be further enhanced
by modulating their functional characteristics, such as
antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), serum half-life,
biodistribution and binding to Fc receptors or the combination of
any of these. This modulation can be achieved by
protein-engineering, glyco-engineering or chemical methods.
Depending on the therapeutic application required, it could be
advantageous to either increase or decrease any of these
activities.
[0138] An example of glyco-engineering used the Potelligent.RTM.
method as described in Shinkawa T. et al., 2003 (J Biol Chem 278:
3466-73).
[0139] Numerous methods for affinity maturation of antibodies are
known in the art. Many of these are based on the general strategy
of generating panels or libraries of variant proteins by
mutagenesis followed by selection and/or screening for improved
affinity. Mutagenesis is often performed at the DNA level, for
example by error prone PCR (Thie, Voedisch et al. 2009), by gene
shuffling (Kolkman and Stemmer 2001), by use of mutagenic chemicals
or irradiation, by use of `mutator` strains with error prone
replication machinery (Greener 1996) or by somatic hypermutation
approaches that harness natural affinity maturation machinery
(Peled, Kuang et al. 2008). Mutagenesis can also be performed at
the RNA level, for example by use of Qf3 replicase (Kopsidas,
Roberts et al. 2006). Library-based methods allowing screening for
improved variant proteins can be based on various display
technologies such as phage, yeast, ribosome, bacterial or mammalian
cells, and are well known in the art (Benhar 2007). Affinity
maturation can be achieved by more directed/predictive methods for
example by site-directed mutagenesis or gene synthesis guided by
findings from 3D protein modeling (see for example Queen, Schneider
et al. 1989 or U.S. Pat. No. 6,180,370 or U.S. Pat. No.
5,225,539).
[0140] Methods of increasing ADCC have been described by Ferrara,
Brunker et al. 2006; Li, Sethuraman et al. 2006; Stavenhagen,
Gorlatov et al. 2007; Shields, Namenuk et al. 2001; Shinkawa,
Nakamura et al. 2003; and WO 2008/006554.
[0141] Methods of increasing CDC have been described by Idusogie,
Wong et al. 2001; Dall'Acqua, Cook et al. 2006; Michaelsen, Aase et
al. 1990; Brekke, Bremnes et al. 1993; Tan, Shopes et al. 1990; and
Norderhaug, Brekke et al. 1991.
[0142] References describing methods of increasing ADCC and CDC
include Natsume, In et al. 2008. The disclosure of each of these
references is included herein by cross reference.
[0143] A number of methods for modulating antibody serum half-life
and biodistribution are based on modifying the interaction between
antibody and the neonatal Fc receptor (FcRn), a receptor with a key
role in protecting IgG from catabolism, and maintaining high serum
antibody concentration. Dall'Acqua et al describe substitutions in
the Fc region of IgG1 that enhance binding affinity to FcRn,
thereby increasing serum half-life (Dall'Acqua, Woods et al. 2002)
and further demonstrate enhanced bioavailability and modulation of
ADCC activity with triple substitution of M252Y/S254T/T256E
(Dall'Acqua, Kiener et al. 2006). See also U.S. Pat. Nos.
6,277,375; 6,821,505; and U.S. Pat. No. 7,083,784. Hinton et al
have described constant domain amino acid substitutions at
positions 250 and 428 that confer increased in vivo half-life
(Hinton, Johlfs et al. 2004). (Hinton, Xiong et al. 2006). See also
U.S. Pat. No. 7,217,797. Petkova et al have described constant
domain amino acid substitutions at positions 307, 380 and 434 that
confer increased in vivo half-life (Petkova, Akilesh et al. 2006).
See also Shields et al 2001 and WO 2000/42072. Other examples of
constant domain amino acid substitutions which modulate binding to
Fc receptors and subsequent function mediated by these receptors,
including FcRn binding and serum half-life, are described in U.S
Pat. Application Nos 20090142340; 20090068175 and 20090092599.
[0144] The glycans linked to antibody molecules are known to
influence interactions of antibody with Fc receptors and glycan
receptors and thereby influence antibody activity, including serum
half-life (Kaneko, Nimmerjahn et al. 2006; Jones, Papac et al.
2007; and Kanda, Yamada et al. 2007). Hence, certain glycoforms
that modulate desired antibody activities can confer therapeutic
advantage. Methods for generating engineered glycoforms are known
in the art and include but are not limited to those described in
U.S. Pat. Nos. 6,602,684; 7,326,681; 7,388,081 and WO
2008/006554.
[0145] Extension of half-life by addition of polyethylene glycol
(PEG) has been widely used to extend the serum half-life of
proteins, as reviewed, for example, by Fishburn 2008.
[0146] As will be recognised it is possible to make conservative
amino acid substitutions within the sequences of the current
invention. By "conservative substitution" is meant amino acids
having similar properties. As used in this specification the
following groups of amino acids are to be seen as conservative
substitutions: H, R and K; D, E, N and Q; V, I and L; C and M; S,
T, P, A and G; and F, Y and W.
[0147] The term "cell surface-associated antigen", as used herein,
broadly refers to any antigen expressed on surfaces of cells,
including infectious or foreign cells or viruses.
[0148] In certain aspects of the present invention, the polypeptide
constructs or compositions of the present invention may be used to
treat patients with cancer. Cancers contemplated herein include: a
group of diseases and disorders that are characterized by
uncontrolled cellular growth (e.g. formation of tumor) without any
differentiation of those cells into specialized and different
cells. Such diseases and disorders include ABL1 protooncogene, AIDS
related cancers, acoustic neuroma, acute lymphocytic leukaemia,
acute myeloid leukaemia, adenocystic carcinoma, adrenocortical
cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part
sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma,
ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer,
bone cancers, bowel cancer, brain stem glioma, brain and CNS
tumors, breast cancer, CNS tumors, carcinoid tumors, cervical
cancer, childhood brain tumors, childhood cancer, childhood
leukaemia, childhood soft tissue sarcoma, chondrosarcoma,
choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloid
leukaemia, colorectal cancers, cutaneous T-Cell lymphoma,
dermatofibrosarcoma-protuberans,
desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine
cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's
sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma,
retinoblastoma, fallopian tube cancer, fanconi anemia,
fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal
cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers,
germ cell tumors, gestational-trophoblastic-disease, glioma,
gynaecological cancers, hematological malignancies, hairy cell
leukaemia, head and neck cancer, hepatocellular cancer, hereditary
breast cancer, histiocytosis, Hodgkin's disease, human
papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx
cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma,
kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer,
leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer,
liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,
malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma,
merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer,
multiple endocrine neoplasia, mycosis fungoides, myelodysplastic
syndromes, multiple myeloma, myeloproliferative disorders, nasal
cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma,
neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin
cancer, non-small-cell-lung-cancer-(NSCLC), ocular cancers,
oesophageal cancer, oral cavity cancer, oropharynx cancer,
osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal
cancer, parathyroid cancer, parotid gland cancer, penile cancer,
peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia
vera, prostate cancer, rare-cancers-and-associated-disorders, renal
cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson
syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary
syndrome, skin cancer, small cell lung cancer (SCLC), small
intestine cancer, soft tissue sarcoma, spinal cord tumors,
squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,
testicular cancer, thymus cancer, thyroid cancer,
transitional-cell-cancer-(bladder),
transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic
cancer, urethral cancer, urinary system cancer, uroplakins, uterine
sarcoma, uterus cancer, vaginal cancer, vulva cancer,
Waldenstrom's-macroglobulinemia and Wilms' tumor. In an embodiment
the tumor is selected from a group of multiple myeloma or
non-hodgkin's lymphoma.
[0149] As contemplated for the treatment of cancer, the antibody
portions of the constructs of the present invention may bind to
tumour-associated antigens, i.e., cell surface antigens that are
selectively expressed by cancer cells or over-expressed in cancer
cells relative to most normal cells. There are many
tumour-associated antigens (TAAs) known in the art. Non-limiting
examples of TAAs include enzyme tyrosinase; melanoma antigen GM2;
alphafetoprotein (AFP); carcinoembryonic antigen (CEA); Mucin 1
(MUC1); Human epidermal growth factor receptor (Her2/Neu); T-cell
leukemia/lymphoma 1 (TCL1) oncoprotein. Exemplary TAAs associated
with a number of different cancers are telomerase (hTERT);
prostate-specific membrane antigen (PSMA); urokinase plasminogen
activator and its receptor (uPA/uPAR); vascular endothelial growth
factor and its receptor (VEGF/VEGFR); extracellular matrix
metalloproteinase inducer (EMMPRIN/CD147); epidermal growth factor
(EGFR); platelet-derived growth factor and its receptor
(PDGF/PDGFR) and c-kit (CD117).
[0150] A list of other TAAs is provided in US 2010/0297076, the
disclosure of which is included herein by reference. Of particular
interest are cell surface antigens associated with multiple myeloma
cells, including but not limited to CD38, CD138, CS1, and HM1.24.
In one embodiment an antigen for antibody-attenuated ligand
constructs, for example, an antibody-attenuated interferon
construct, is CD38.
[0151] CD38 is a 46 kDa type II transmembrane glycoprotein. It has
a short N-terminal cytoplasmic tail of 20 amino acids, a single
transmembrane helix and a long extracellular domain of 256 amino
acids (Bergsagel, P., Blood; 85:436, 1995 and Liu, Q., Structure,
13:1331, 2005). It is expressed on the surface of many immune cells
including CD4 and CD8 positive T cells, B cells, NK cells,
monocytes, plasma cells and on a significant proportion of normal
bone marrow precursor cells (Malavasi, F., Hum. Immunol. 9:9,
1984). In lymphocytes, however, the expression appears to be
dependent on the differentiation and activation state of the cell.
Resting T and B cells are negative while immature and activated
lymphocytes are predominantly positive for CD38 expression (Funaro,
A., J. Immunol. 145:2390, 1990). Additional studies indicate mRNA
expression in non-hemopoeitic organs such as pancreas, brain,
spleen and liver (Koguma, T., Biochim. Biophys. Acta 1223:160,
1994.)
[0152] CD38 is a multifunctional ectoenzyme that is involved in
transmembrane signaling and cell adhesion. It is also known as
cyclic ADP ribose hydrolase because it can transform NAD.sup.+ and
NADP.sup.+ into cADPR, ADPR and NAADP, depending on extracellular
pH. These products induce Ca.sup.2+-mobilization inside the cell
which can lead to tyrosine phosphorylation and activation of the
cell. CD38 is also a receptor that can interact with a ligand,
CD31. Activation of receptor via CD31 leads to intracellular events
including Ca.sup.2+ mobilization, cell activation, proliferation,
differentiation and migration (reviewed in Deaglio, S., Trends in
Mol. Med. 14:210, 2008.)
[0153] CD38 is expressed at high levels on multiple myeloma cells,
in most cases of T- and B-lineage acute lymphoblastic leukemias,
some acute myelocytic leukemias, follicular center cell lymphomas
and T lymphoblastic lymphomas. (Malavasi, F., J. Clin Lab Res.
22:73, 1992). More recently, CD38 expression has become a reliable
prognostic marker in B-lineage chronic lymphoblastic leukemia
(B-CLL) (Ibrahim, S., Blood. 98:181, 2001 and Durig, J., Leuk. Res.
25:927, 2002). Independent groups have demonstrated that B-CLL
patients presenting with a CD38.sup.+ clone are characterized by an
unfavorable clinical course with a more advance stage of disease,
poor responsiveness to chemotherapy and shorter survival time
(Morabito, F., Haematologica. 87:217, 2002). The consistent and
enhanced expression of CD38 on lymphoid tumors makes this an
attractive target for therapeutic antibody technologies.
[0154] Preferred antigens for the development of
antibody-attenuated ligand fusion protein constructs which target
cancer are antigens which show selective or greater expression on
the cancer cells than on most other, non-transformed cells within
the body. Non-protein examples of such antigens include,
sphingolipids, ganglioside GD2 (Saleh et al., 1993, J. Immunol.,
151, 3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer
Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et
al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et
al., 1993, Cancer Res. 53:5244-5250) and Lewis.sup.x, lewis.sup.y
and lewis.sup.xy carbohydrate antigens that can be displayed on
proteins or glycolipids. Examples of protein antigens are
HER-2/neu, human papillomavirus-E6 or -E7, MUC-1; KS 1/4
pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol.
142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian
carcinoma antigen CA125 (Yu et al., 1991, Cancer Res.
51(2):468-475); prostatic acid phosphate (Tailor et al., 1990,
Nucl. Acids Res. 18(16):4928); prostate specific antigen (Henttu
and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910;
Israeli et al., 1993, Cancer Res. 53:227-230); melanoma-associated
antigen p97 (Estin et al., 1989, J. Natl. Cancer Instit.
81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al., 1990,
J. Exp. Med. 171(4):1375-1380); prostate specific membrane antigen;
carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc.
Clin. Oncol. 13:294), MUC16 (antibodies include MJ-170, MJ-171,
MJ-172 and MJ-173 [U.S. Pat. No. 7,202,346], 3A5 [U.S. Pat. No.
7,723,485]). NMB (U.S. Pat. No. 8,039,593), malignant human
lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science
245:301-304); high molecular weight melanoma antigen (BMW-MAA)
(Natali et al., 1987, Cancer 59:55-63; Mittelman et al., 1990, J.
Clin. Invest. 86:2136-2144); Burkitt's lymphoma antigen-38.13; CD19
(Ghetie et al., 1994, Blood 83:1329-1336); human B-lymphoma
antigen-CD20 (Reff et al., 1994, Blood 83:435-445); GICA 19-9
(Herlyn et al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA; CD33
(Sgouros et al., 1993, J. Nucl. Med. 34:422-430); oncofetal
antigens such as alpha-fetoprotein for liver cancer or bladder
tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res.
45:2210-2188); differentiation antigens such as human lung
carcinoma antigen L6 or L20 (Hellstrom et al., 1986, Cancer Res.
46:3917-3923); antigens of fibrosarcoma; human leukemia T cell
antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immunol.
141:1398-1403); tumor-specific transplantation type of cell-surface
antigen (TSTA) such as virally-induced tumor antigens including
T-antigen, DNA tumor virus and envelope antigens of RNA tumor
viruses; neoglycoproteins, breast cancer antigens such as EGFR
(Epidermal growth factor receptor), polymorphic epithelial mucin
(PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359);
polymorphic epithelial mucin antigen; human milk fat globule
antigen; colorectal tumor-associated antigens such as TAG-72
(Yokata et al., 1992, Cancer Res. 52:3402-3408), CO 17-1A
(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758);
differentiation antigens (Feizi, 1985, Nature 314:53-57) such as
I(Ma) found in gastric adenocarcinomas, SSEA-1 found in myeloid
cells, VEP8, VEP9, Myl, VIM-D5, M18 and M39 found in breast
epithelial cancers, D.sub.156-22 found in colorectal cancer,
TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3
found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten
found in embryonal carcinoma cells, TL5 (blood group A), E1 series
(blood group B) antigens found in pancreatic cancer, FC10.2 found
in embryonal carcinoma cells, gastric adenocarcinoma antigen,
CO-514 (blood group Le.sup.a) found in adenocarcinoma, NS-10 found
in adenocarcinomas, CO-43 (blood group Le.sup.b), G49 found in A431
cells, 19.9 found in colon cancer; gastric cancer mucins; R.sub.24
found in melanoma, MH2 (blood group ALe.sup.b/LeY) found in colonic
adenocarcinoma, 4.2, D1.1, OFA-1, G.sub.M2, OFA-2 and M1:22:25:8
found in embryonal carcinoma cells and SSEA-3 and SSEA-4. HMW-MAA
(SEQ ID NO:433), also known as melanoma chondroitin sulfate
proteoglycan, is a membrane-bound protein of 2322 residues which is
overexpressed on over 90% of the surgically removed benign nevi and
melanoma lesions (Camploi, et. al, Crit Rev Immunol.; 24:267,
2004). Accordingly it may be a potential target cell surface
associated antigen.
[0155] Other example cancer antigens for targeting with fusion
protein constructs of the present invention include (exemplary
cancers are shown in parentheses): CD5 (T-cell leukemia/lymphoma),
CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), CA 242
(colorectal), placental alkaline phosphatase (carcinomas),
prostatic acid phosphatase (prostate), MAGE-1 (carcinomas), MAGE-2
(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), transferrin
receptor (carcinomas), p97 (melanoma), MUC1 (breast cancer), MARTI
(melanoma), CD20 (non Hodgkin's lymphoma), CD52 (leukemia), CD33
(leukemia), human chorionic gonadotropin (carcinoma), CD38
(multiple myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), CD25
(B-cell Lymphoma), CD37 (B-cell lymphoma), CD45 (acute myeloblastic
leukemia), HLA-DR (B-cell lymphoma), IL-2 receptor (T-cell leukemia
and lymphomas), CD40 (lymphoma), various mucins (carcinomas), P21
(carcinomas), MPG (melanoma), Ep-CAM (Epithelial Tumors),
Folate-receptor alpha (Ovarian), A33 (Colorectal), G250 (renal),
Ferritin (Hodgkin lymphoma), de2-7 EGFR (glioblastoma, breast, and
lung), Fibroblast activation protein (epithelial) and tenascin
metalloproteinases (glioblastoma). Some specific, useful antibodies
include, but are not limited to, BR64 (Trail et al., 1997, Cancer
Research 57:100 105), BR96 mAb (Trail et al., 1993, Science
261:212-215), mAbs against the CD40 antigen, such as S2C6 mAb
(Francisco et al., 2000, Cancer Res. 60:3225-3231) or other
anti-CD40 antibodies, such as those disclosed in U.S Patent
Publication Nos. 2003-0211100 and 2002-0142358; mAbs against the
CD30 antigen, such as AC10 (Bowen et al., 1993, J. Immunol.
151:5896-5906; Wahl et al., 2002 Cancer Res. 62(13):3736-42) or
MDX-0060 (U.S. Patent Publication No. 2004-0006215) and mAbs
against the CD70 antigen, such as 1F6 mAb and 2F2 mAb (see, e.g.,
U.S. Patent Publication No. 2006-0083736) or antibodies 2H5, 10B4,
8B5, 18E7, 69A7 (U.S. Pat. No. 8,124,738). Other antibodies have
been reviewed elsewhere (Franke et al., 2000, Cancer Biother.
Radiopharm. 15:459 76; Murray, 2000, Semin. Oncol. 27:64 70;
Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley,
and Sons, New York, 1998).
[0156] In certain embodiments, useful antibodies can bind to a
receptor or a complex of receptors expressed on a target cell. The
receptor or receptor complex can comprise an immunoglobulin gene
superfamily member, a major histocompatibility protein, a cytokine
receptor, a TNF receptor superfamily member, a chemokine receptor,
an integrin, a lectin, a complement control protein, a growth
factor receptor, a hormone receptor or a neuro-transmitter
receptor. Non-limiting examples of appropriate immunoglobulin
superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD79, CD90,
CD152/CTLA-4, PD-1, B7-H4, B7-H3, and ICOS. Non-limiting examples
of suitable TNF receptor superfamily members are TACI, BCMA, CD27,
CD40, CD95/Fas, CD134/0X40, CD137/4-1BB, TNFR1, TNFR2, RANK,
osteoprotegerin, APO 3, Apo2/TRAIL R1, TRAIL R2, TRAIL R3, and
TRAIL R4. Non-limiting examples of suitable integrins are CD11a,
CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e,
CD49f, CD103 and CD104. Non-limiting examples of suitable lectins
are S type, C type, and I type lectin. Examples of antibodies to
CEA are shown in Table 2.
TABLE-US-00003 TABLE 2 CEA Antibodies Ab Clones Patent Assignee
Comments COL-1 U.S. Pat. No. 6,417,337 The Dow Chemical Humanized
Company 806.077 U.S. Pat. No. 6,903,203 AstraZeneca UK Ltd.
Humanized T84.66 U.S. Pat. No. 7,776,330 City of Hope Humanized
[0157] Antibodies that bind the CD22 antigen expressed on human B
cells include, for example, HD6, RFB4, UV22-2, To15, 4KB128 and a
humanized anti-CD22 antibody (hLL2) (see, e.g., Li et al. (1989)
Cell. Immunol. 111: 85-99; Mason et al. (1987) Blood 69: 836-40;
Behr et al. (1999) Clin. Cancer Res. 5: 3304s-3314s; Bonardi et al.
(1993) Cancer Res. 53: 3015-3021).
[0158] Antibodies to CD33 include, for example, HuM195 (see, e.g.,
Kossman et al. (1999) Clin. Cancer Res. 5: 2748-2755; U.S. Pat. No.
5,693,761) and CMA-676 (see, e.g., Sievers et al., (1999) Blood 93:
3678-3684).
[0159] Illustrative anti-MUC-1 antibodies include, but are not
limited to Mc5 (see, e.g., Peterson et al. (1997) Cancer Res. 57:
1103-1108; Ozzello et al. (1993) Breast Cancer Res. Treat. 25:
265-276), and hCTMO1 (see, e.g., Van Hof et al. (1996) Cancer Res.
56: 5179-5185).
[0160] Illustrative anti-TAG-72 antibodies include, but are not
limited to CC49 (see, e.g., Pavlinkova et al. (1999) Clin. Cancer
Res. 5: 2613-2619), B72.3 (see, e.g., Divgi et al. (1994) Nucl.
Med. Biol. 21: 9-15), and those disclosed in U.S. Pat. No.
5,976,531.
[0161] Illustrative anti-HM1.24 antibodies include, but are not
limited to a mouse monoclonal anti-HM1.24 and a humanized
anti-HM1.24 IgGlkappa antibody (see, e.g., Ono et al. (1999) Mol.
Immuno. 36: 387-395).
[0162] In certain embodiments the targeting moiety comprises an
anti-HER2 antibody. The erBB 2 gene, more commonly known as
(Her-2/neu), is an oncogene encoding a transmembrane receptor.
Several antibodies have been developed against Her-2/neu, including
trastuzumab (e.g., HERCEPTIN.TM.; Fornier et al. (1999) Oncology
(Huntingt) 13: 647-58), TAB-250 (Rosenblum et al. (1999) Clin.
Cancer Res. 5: 865-874), BACH-250 (Id.), TA1 (Maier et al. (1991)
Cancer Res. 51: 5361-5369), and the mAbs described in U.S. Pat.
Nos. 5,772,997; 5,770,195 (mAb 4D5; ATCC CRL 10463); and U.S. Pat.
No. 5,677,171.
[0163] A number of antibodies have been developed that specifically
bind HER2 and some are in clinical use. These include, for example,
trastuzumab (e.g., HERCEPTIN.TM.., Fornier et al. (1999) Oncology
(Huntingt) 13: 647-658), TAB-250 (Rosenblum et al. (1999) Clin.
Cancer Res. 5: 865-874), BACH-250 (Id.), TA1 (see, e.g., Maier et
al. (1991) Cancer Res. 51: 5361-5369), and the antibodies described
in U.S. Pat. Nos. 5,772,997; 5,770,195, and 5,677,171.
[0164] Other fully human anti-HER2/neu antibodies are well known to
those of skill in the art. Such antibodies include, but are not
limited to the C6 antibodies such as C6.5, DPL5, G98A, C6MH3-B1,
B1D2, C6VLB, C6VLD, C6VLE, C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5,
C6MH3-D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, C6MH3-C3, C6MH3-B9,
C6MH3-B5, C6MH3-B48, C6MH3-B47, C6MH3-B46, C6MH3-B43, C6MH3-B41,
C6MH3-B39, C6MH3-B34, C6MH3-B33, C6MH3-B31, C6MH3-B27, C6MH3-B25,
C6MH3-B21, C6MH3-B20, C6MH3-B2, C6MH3-B16, C6MH3-B15, C6MH3-B11,
C6MH3-B1, C6MH3-A3, C6MH3-A2, and C6ML3-9. These and other
anti-HER2/neu antibodies are described in U.S. Pat. Nos. 6,512,097
and 5,977,322, in PCT Publication WO 97/00271, in Schier et al.
(1996) J Mol Biol 255: 28-43, Schier et al. (1996) J Mol Biol 263:
551-567, and the like.
[0165] More generally, antibodies directed to various members of
the epidermal growth factor receptor family are well suited for use
as targeting antibodies or antigen binding portions thereof in the
constructs of the present invention. Such antibodies include, but
are not limited to anti-EGF-R antibodies as described in U.S. Pat.
Nos. 5,844,093 and 5,558,864, and in European Patent No. 706,799A.
Other illustrative anti-EGFR family antibodies include, but are not
limited to antibodies such as C6.5, C6ML3-9, C6MH3-B1, C6-B1D2, F5,
HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12,
EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8,
HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7,
HER4.F8 and HER4.C7 and the like (see, e.g., U.S. Patent
publications US 2006/0099205 A1 and US 2004/0071696 A1 which are
incorporated herein by reference).
[0166] It may be advantageous for the cell surface-associated
antigen to be expressed at sufficient levels on the target cell
that a sufficently therapeutic amount of polypeptide construct is
presented to ligand receptors on the target cell surface.
Accordingly, in particular embodiments, the cell surface associated
antigen is expressed at a density of greater than 12,600 copies per
cell or greater than 15,000 copies per cell. Methods for
determining copy number of a cell surface antigen are well known
and readily available to a person of skill in the art, for example
the method provided by Jilana (Am J Clin Pathol 118:560-566,
2002)
[0167] It may be advantageous for the cell surface-associated
antigen to be expressed in a configuration on the cell surface such
that the polypeptide construct is abe to contact both the cell
surface antigen and the ligand receptor on the target cell.
Accordingly, in particular embodiments the cell surface associated
antigen has an extracelluar domain having a molecular weight of
less than 240 kD.
[0168] It may be advantageous for the antibody or antigen-binding
portion thereof to bind to the cell surface associated antigen with
sufficient affinity to facilitate ligand binding to the ligand
receptor on the cell surface. Accordingly, in particular
embodiments of the the present invention the polypeptide constructs
exhibit an antigen binding affinity, as measured by EC50, of from
50 nM, from 25 nM, from 10 nM or from 5 nM to 0.1 pM.
[0169] As described in U.S. Pat. Nos. 6,512,097 and 5,977,322,
other anti-EGFR family member antibodies can readily be produced by
shuffling light and/or heavy chains followed by one or more rounds
of affinity selection. Thus in certain embodiments, this invention
contemplates the use of one, two, or three CDRs in the VL and/or VH
region that are CDRs described in the above-identified antibodies
and/or the above identified publications.
[0170] In various embodiments the targeting antibody or antigen
binding portion thereof comprises an antibody or antigen binding
portion thereof that specifically or preferentially binds CD20.
Anti-CD20 antibodies are well known to those of skill and include,
but are not limited to Rituximab, Ibritumomab, and Tositumomab,
AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche),
Ofatumumab (Genmab), TRU-015 (Trubion) and IMMU-106
(Immunomedics).
[0171] WO 2010/105290 discloses an antibody designated "SC104"
together with a range of humanised variants which bind an antigen
expressed on a range of tumour cells.
[0172] In an embodiment, the antibody attachment and attenuating
mutation in the ligand increases the antigen-specificity index
(ASI) by greater than 10-fold, preferably greater than 50-fold,
preferably greater than 100-fold, preferably greater than
1000-fold, or preferably greater than 10,000 fold. The
antigen-specificity index (ASI) is defined herein as the fold
increased potency in signaling activity of the polypeptide
construct of the invention relative to the free non-mutated
polypeptide ligand on target antigen-positive cells multiplied by
the fold decreased potency in signaling activity relative to the
free non-mutated polypeptide ligand on target antigen-negative
cells. The term "potency" in this context may be quantitatively
represented by the EC50 value, which is the mathematical midpoint
of a dose-response curve, in which the dose refers to the
concentration of ligand or antibody-ligand construct in an assay,
and response refers to the quantitative response of the cells to
the signaling activity of the ligand at a particular dose. Thus,
for example, when a first compound is shown to possess an EC50
(expressed for example in Molar units) that is 10-fold lower than a
second compound's EC50 on the same cells, typically when measured
by the same method, the first compound is said to have a 10-fold
higher potency. Conversely, when a first compound is shown to
possess an EC50 that is 10-fold higher than a second compound's
EC50 on the same cells, typically when measured by the same method,
the first compound is said to have a 10-fold lower potency.
[0173] While the large majority of antibodies tested showed
efficient targeting of attenuated IFN.alpha. the present inventors
identified examples of two antigens where targeting attenuated
IFN.alpha. to a target-expressing cell line did not exhibit an ASI
that was appreciably greater than for the free, non-mutated ligand.
The first example is demonstrated by the antigen CSPG4 (also known
as BMW-MAA, high molecular weight melanoma-associated antigen). We
tested two different anti-HMW-MAA-antibody-IFN.alpha. fusion
protein constructs in on-target proliferation assays using A375 or
CHL-1 cell lines. We did not see inhibitory activity with either
cell line or antibody at the doses tested (EC50s>21 nM). The
extracelluar domain of this antigen is exceptionally large
(extracellular domain MW approx. 240 kD-450 kD depending on
glycosylation). It is possible that certain antibody-IFN fusion
protein constructs that bind to very large antigens may be
sterically restricted from simultaneously interacting with the IFN
receptors on the same cells. It is, however, possible that other
antibodies that target other epitopes of this antigen may support
the targeted IFN activity. Despite this possibility it is preferred
that the antibody or antigen binding portion thereof of the
polypeptide construct of the present invention binds an antigen
wherein the extracellular domain thereof has a molecular weight of
less than 240 kD.
[0174] A second example of an antibody-attenuated IFN.alpha. fusion
protein construct that did not show potent activity was based on an
antibody which binds to the myeloid antigen CD33. CD33 is expressed
at a relatively low level on KG-1 cells, reported as 12,600 copies
per cell (Sutherland, MAbs. 1(5): 481-490, 2009). Treatment of KG-1
cells with an anti-CD33 antibody-attenuated IFN.alpha. fusion
protein construct failed to inhibit proliferation at all doses
tested (IC50>76 nM). We believe that the relatively low copy
number of this target may in some cases, depending on other factors
such as epitope position, the receptor density of the IFN
receptors, etc, limit the potency of the antibody-attenuated IFN
fusion protein constructs. It is, however, possible that other
antibodies that target other epitopes on this antigen may support
the targeted IFN activity, or that other cells with low copy
numbers of CD33 may nevertheless respond to such fusion protein
constructs due to higher intrinsic IFN sensitivity, for example.
Despite this possibility it is preferred that the antibody or
antigen binding portion thereof of the polypeptide construct of the
present invention binds an antigen wherein the antigen is present
on the cell at a density of greater than 12,600 copies per cell,
preferably greater than 15,000 copies per cell.
[0175] Another example of an antibody-attenuated fusion protein
construct in which the antibody did not provide sufficient
targeting to the cancer cells was an anti-GM2 ganglioside antibody
attached to an attenuated IFN.alpha.. In this case, the antibody
was to a carbohydrate epitope and, as typical of such antibodies,
had a low affinity (EC50 for binding target cells was .about.50 nM
by flow cytometry). Therefore, preferred embodiments of the present
invention show high affinity binding to their antigens, with EC50s
preferably below 50 nM, more preferably below 25 nM, still more
preferably below 10 nM and ideally below 5 nM. In addition,
preferred embodiments comprise antibodies that bind to protein and
peptide epitopes rather than carbohydrate epitopes.
[0176] Multiple myeloma is of particular interest for certain
embodiments of the present invention, namely fusion protein
constructs comprising antibodies to multiple myeloma antigens and
attenuated IFN peptides. Table 3 lists examples of multiple myeloma
antigens and antibodies, with a reference to antibody
sequences.
TABLE-US-00004 TABLE 3 Examples of Ab in preclinical or clinical
Clinical trial Target development Sequence citation reference CD40
Dacetuzumab SGN-40 USPTO Granted NCT00664898 & Patent
#7,666,422 NCT00525447 CD40 Lucatumumab HCD-122 USPTO#20070098718
NCT00231166 CHIR12.12 HM1.24 XmAb5592 humanized + Fc
USPTO#20100104557 1999, Ozaki, Blood, 93: 3922 CD56 HuN901-DM1
1994, Roguska et al., NCT00346255 & BB-10901 PNAS 91: 969-973
NCT00991562 CS1 Elotuzumab HuLuc63 USPTO Granted NCT00742560 Patent
#7,709,610 &NCT00726869 CD138 nBT062 USPTO #20090175863 2008,
Tassone, Blood, 104: 3688 CD74 Milatuzumab Immu-110 US. Granted
Patent # NCT00421525, Stein 7,312,318 et. Al. 2007 and 2009 IL-6R
Tocilizumab MRA US Granted Patent 2007, Yoshio- #5,795,965 Hoshino,
Canc Res, 67; 871 Trail-R1 Mapatumumab, anti-DR4 US Granted Patent
# NCT00315757 7,252,994 Trail-R2 (DR5, Lexatumumab, ETR2-ST01, US
Granted Patent # 2006, Menoret, APO-2) anti-DR5 6,872,568 Blood,
132; 1356 Baff Belimumab LY2127399 US Granted Patent # 7,317,089
ICOSL AMG-557 USPTO Application Number 20080166352 BCMA SG1 USPTO
Application 2007, Ryan, Mol Number 2012008266 Cancer Ther, 6: 3009
HLA-DR 1D09C3 USPTO Granted 2007, Carlo-Stella, Pantent # 7,521,047
Canc. Res., Kininogen C11C1 USPTO Granted 2006, Sainz, Canc Patent
# 4,908,431 Immunol Immunother .beta.2microglobulin ATCC Cat
#HB-149 2007, Yang, Blood, 110: 3028; 2009, Clin Can Res, 15: 951
FGFR3 Pro-001 USPTO Granted 2006, Trudel, Blood, Patent # 8,187,601
2: 4908 ICAM-1 cUV3 USPTO Granted 2004, Smallshaw, J Patent #
7,943,744 Immunother; 2006, Coleman Matriptase M24-DOX USPTO
Granted 2010, Bertino, AACR Patent #7,355,015 abstract no. 2596
CD20 Rituxan and others U.S. Patent NCT00258206 & Application
Number: NCT00505895 US 2010/0189729 A1 CD52 Campath-1H USPTO
Granted NCT00625144 Patent #6,569,430 EGFR Erbitux (Emma-1) USPTO
Granted NCT00368121 Patent #6,217,866 GM2 BIW-8962 USPTO Granted
Biowa, no ref Patent # 6,872,392 .alpha.4-integrin natalizumab
USPTO Granted NCT00675428 Patent # 5,840,299 IGF-1R CD-751,871
figitumumab USPTO Granted Lacy, J. Clin. Oncol, Patent # 7,700,742
26: 3196 (TBD - need to connect Ab 4.9.2 to CD751,871) KIR IPH2101
USPTO Granted NCT00552396; Patent # 8,119,775 2009, ASCO abs 09-
AB-3032;
[0177] CD38 is of particular interest as an antibody target for
fusion protein constructs of the present invention. Antibodies to
CD38 include for example, AT13/5 (see, e.g., Ellis et al. (1995) J.
Immunol. 155: 925-937), HB7, and the like. Table 4 discloses
several known CD38 antibodies that may be used in this context:
TABLE-US-00005 TABLE 4 Company Clone names Sequence citation Ref
Genmab/ G003. G005, G024 WO 2006/099875 A1 De Weers, M., J Janssen
Biotech (Daratumumab) Immunol. Inc 186: 1840, 2011 MorphoSys AG
MOR03077, US 2009/0123950 A1 MORO3079, MORO3080, MORO3100 (MOR202)
Sanofi-Aventis 38SB13, 38SB18, US 2009/0304710 A1 US. LLC. 38SB19,
38SB30, 38SB31, 38SB39 (SAR650984) Tenovus UK Chimeric OKT10 US
2010/0285004 A1 Stevenson, F., Parental hybridoma Blood. 77: 1071,
ATCC accession: CRL- 1991 8022 Immunogen HB7-Ricin Hybridoma: ATCC
HB- Goldmacher, V., 136 Blood, 84: 3017, 1994
[0178] The term "Signaling ligand" as used herein broadly includes
any ligand involved in the activation of cell signaling pathways,
including any molecule capable of activating or inhibiting cell
surface receptors. The term should also be understood as including
reference to molecules that can pass through the lipid bilayer of
the cell membrane to activate cell signaling pathways within the
cell. The term "polypeptide signaling ligand" as used herein refers
to peptide and polypeptide sequences of length 6 amino acids
through 1,000 amino acids in length, which bind to particular cell
surface molecules ("receptors") on certain cells and thereby
transmit a signal or signals within those cells. Exemplary
signaling ligands and polypeptide signaling ligands contemplated by
the present invention include, but are not limited to cytokines,
chemokines, growth factors, hormones, neurotransmitters, and
apoptosis inducing factors.
[0179] Non-limiting examples of suitable cytokines include the
interleukin's IL-1, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL_25, IL-26, IL-27,
IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, 11-35 and their
subfamiles; the interferon (IFN) subfamily including Interferon
type I (IFN-.alpha. (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7,
IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21), IFN-.beta.
(IFN-.beta.1 (IFNB1) and IFN-.beta.3 (IFNB3)), IFN-.omega.
((IFNW1), IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and
IFNWP19 and IFNK), Interferon type II (IFN-.gamma.) and Interferon
type III (IFN-epsilon, -kappa, -omega, -delta, -tau, and -gamma)
and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29;
the IL-1 family including IL-1.alpha., IL-1.beta., the IL-1
Receptor antagonist (IL-1RA) and IL1F5, IL1F6, IL1F7, IL1F8, IL1F9
and IL1F10 and the IL-17 family including IL-17A, IL-17B, IL-17C,
IL-17D, IL-17E (IL-25), and IL-17F. In an embodiment the peptide or
polypeptide signaling ligand is selected from the group consisting
of an IFN, IL-4 and IL-6. In an embodiment the peptide or
polypeptide signaling ligand is selected from the group consisting
of IFN.alpha., IFN.alpha.2b, IFN.beta.1, IFN.beta.1b and
IFN.gamma.. Preferably the sequence of IFN.alpha. is selected from
SEQ ID NOs 1 to 3, 80 to 90, 434 and 435.
[0180] Exemplary chemokines include, for example, RANTES, MCAF,
MIP1-alpha, IP-10, monocyte chemoattractant protein-1 (MCP-1 or
CCL2), interleukin-8 (IL-8), CXCL13, XCL1 (lymphotactin-a), XCL2
(lymphotactin-3) and fractalkine (CX.sub.3CL1).
[0181] Non-limiting examples of growth factors include, for
example, Adrenomedullin (AM), Angiopoietin (Ang), Autocrine
motility factor, Bone morphogenetic proteins (BMPs), Brain-derived
neurotrophic factor (BDNF), Epidermal growth factor (EGF),
Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell
line-derived neurotrophic factor (GDNF), Granulocyte
colony-stimulating factor (G-CSF), Granulocyte macrophage
colony-stimulating factor (GM-CSF), Growth differentiation factor-9
(GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth
factor (HDGF), Insulin-like growth factor (IGF),
Migration-stimulating factor, Myostatin (GDF-8), Nerve growth
factor (NGF) and other neurotrophins, Platelet-derived growth
factor (PDGF), Thrombopoietin (TPO), Transforming growth factor
alpha (TGF-.alpha.), Transforming growth factor beta (TGF-.beta.),
Tumor_necrosis_factor-alpha (TNF-.alpha.), Vascular endothelial
growth factor (VEGF), placental growth factor (P1GF), IL-1-Cofactor
for IL-3 and IL-6, IL-2-T-cell growth factor, IL-3, IL-4, IL-5,
IL-6 and IL-7.
[0182] Exemplary apoptosis inducing factors include FasL and
TRAIL.
[0183] Exemplary hormones include peptide hormones such as TRH and
vasopressin, protein hormones such as insulin and growth hormone,
glycoprotein hormones such as Luteinizing hormone,
follicle-stimulating hormone and thyroid-stimulating hormone, Lipid
and phospholipid-derived hormones such as steroid hormones e.g.
testosterone and cortisol, Sterol hormones such as calcitriol,
eicosanoids such as prostaglandins.
[0184] Non-limiting examples of suitable neurotransmitters
includemonoamines and other biogenic amines: dopamine (DA),
norepinephrine (noradrenaline; NE, NA), epinephrine (adrenaline),
histamine, serotonin (SE, 5-HT), somatostatin, substance P, opioid
peptides and acetylcholine (ACh),
[0185] The linkage between the antibody and the ligand could be
made via a simple peptide bond by creating a fusion protein between
the ligand and the heavy or light chain, or both, of the antibody.
The ligand could be attached at either the N- or C-terminus of
either the heavy or the light chain of the antibody, with or
without an intervening linker peptide sequence. In an embodiment
the ligand is linked to the antibody or antigen binding portion
thereof via a peptide bond. In one embodiment, the ligand is linked
to the C-terminus of the heavy chain of a human, humanized or
chimeric IgG1, IgG2 or IgG4, either directly or with an intervening
linker of 1 to 20 amino acids in length.
[0186] The mutated polypeptide ligands may be attached to the
antibody or antibody fragment by means of chemical conjugation,
non-covalent protein-protein interactions, or by genetic fusion.
Methods for conjugating the ligands described herein with
antibodies may be readily accomplished by one of ordinary skill in
the art. As will be readily ascertained, commonly used chemical
coupling methods may be utilized to link ligands to antibodies via
for example, free amino, carboxylic acid, or sulfhydryl groups.
Ligands can also be linked to antibodies via Carbonyls (--CHO);
these aldehyde groups can be created by oxidizing carbohydrate
groups in glycoproteins.
[0187] Some commonly used cross-linking reagents include
glutaraldehyde which links protein or peptide molecules to the
N-terminal or aliphatic amine groups of peptides or polypeptides,
carbodiimide (EDC) which attaches proteins or peptides to the
C-terminus or side chain carboxyl groups of proteins or peptides,
succinimide esters (e.g. MBS, SMCC) which conjugates free amino
groups and thiols from Cys residues, benzidine (BDB) which links to
Tyr residues, periodate which attaches to carbohydrate groups and
isothiocyanate. The use of commercial chemical conjugation kits is
contemplated.
[0188] In some embodiments, labels are attached via spacer arms of
various lengths to reduce potential steric hindrance. For example,
a chemical linker may be used between the ligand and the antibody.
Exemplary linker sequences will be readily ascertained by those of
skill in the art, and are likely to include linkers such as C6, C7
and C12 amino modifiers and linkers comprising thiol groups.
[0189] The antibody-ligand fusion protein constructs of the present
invention have mutations or deletions in the ligand that render the
ligands less active in stimulating their receptors on cells that
lack cell surface expression of the antigen to which the antibody
binds.
[0190] In one aspect of the present invention, the ligand is an
interferon, examples of which are type I interferons (IFN-.alpha.
(alpha), IFN-.beta. (beta), IFN-.kappa. (kappa), IFN-.delta.
(delta), IFN-.epsilon. (epsilon), IFN-.tau. (tau), IFN-.omega.
(omega), and IFN-.zeta. (zeta, also known as limitin), type II
interferons (IFN-.gamma.) or type III interferons (IFN-.lamda.1,
IFN-.lamda.2 and IFN-.lamda.3) (Pestka, Immunological Reviews
202(1):8-32, 2004).
[0191] Type I interferons all signal through the Type I interferon
receptor, which is made of IFNAR1 and IFNAR2. Signaling occurs when
a type I IFN binds IFNAR1 and IFNAR2, thus bringing them together
into a complex with the IFN. This initiates a cascade of
intracellular events (the "signaling") which leads, among other
things, to changes in the expression of numerous interferon
regulated genes. Details of the intracellular signaling events
triggered by activation of the type I interferon receptor is
described, for example, by Platanias, (Nature Reviews 5:375-86.
2005). Type I interferons include various interferon-alphas. Known
human interferon-alphas are
[0192] IFN.alpha.1b, .alpha.2.alpha., .alpha.2.beta., .alpha.4b,
.alpha.5, .alpha.6, .alpha.7, .alpha.8, .alpha.10, .alpha.1a/13,
.alpha.14, .alpha.16, .alpha.17, .alpha.v.delta. .alpha.21,
.alpha.2c and .alpha.4a. Some embodiments comprise IFN.alpha.2b,
the sequence of which, SEQ ID NO:3, is shown in FIG. 4a. IFNs have
been approved in several forms for several indications, as outlined
in Table 5 (which also shows lists of approved IFN.beta. and
.gamma.'s):
TABLE-US-00006 TABLE 5 Generic Name Trade name Approved for
treatment Interferon alpha 2a ROFERON .RTM. A (Hoffman-La Hep C,
CML, Hairy cell Roche Inc., Nutley, NJ) Leukemia, NHL, Kaposi's
sarcoma Interferon alpha 2b Intron A/Reliferon/Uniferon Hep C, Hep
B, Hairy cell, melanoma, leukemia, NHL, Kaposi's sarcoma Human
leukocyte Interferon MULTIFERON .RTM. (Viranative AB, Melanoma,
viral and (HuIFN- Le) Umea Sweden) malignant disease Interferon
beta 1a, liquid REBIF .RTM. (Ares Trading Multiple Sclerosis S.A.,
Aubonne Switzerland) Interferon beta 1a, AVONEX .RTM. (Biogen,
Inc., Multiple Sclerosis lyophylized Cambridge, MA) Interferon beta
1a, Cinnovex Multiple Sclerosis biogeneric (Iran) Interferon beta
1b BETASERON .RTM./Betaferon (Bayer Multiple Sclerosis Pharma
Aktiengesellshaft, Berlin Germany) Interferon beta 1b, Ziferon
Multiple Sclerosis biosimilar (Iran) PEGylated interferon alpha
PEGASYS .RTM. (Hoffman-La Roche Hepatitis B and C 2a Inc., Nutley,
NJ) PEGylated interferon alpha Reiferon Retard Hep C, Hep B, Hairy
cell, 2a (Egypt) melanoma, leukemia, NHL, Kaposi's sarcoma
PEGylated interferon alpha PEGINTRON .RTM. (Merck Sharpe &
Hepatitis and melanoma 2b Dome Corp., Kenilworth, NJ) PEGylated
interferon alpha Pegetron Hepatitis C 2b plus ribavirin (Canada)
Interferon alfacon-1 INFERGEN .RTM. (Amgen, Inc., Hepatitis C
Thousand Oaks, CA) Interferon alpha n3 ALFERON N .RTM. (Hemispherx
Genital warts Biopharma, Inc., Philadelphia, PA) Interferon gamma
ACTIMMUNE .RTM. (Genentech, Chronic granulomatous Inc., San
Francisco, CA) disease
[0193] Non-limiting examples of mutations in IFN.alpha.2b that can
be used to reduce its potency are described in Tables 6 and 7,
based on the sequence of human IFN.alpha.2b (SEQ ID NO:3):
TABLE-US-00007 TABLE 6 Relative biological activities of interferon
mutants relative anti- relative anti-viral proliferative activity
activity IFN.alpha.2b wild type 1 1 L15A 0.079 0.29 R22A 0.9 R23A
0.4 0.49 S25A 0.76 0.7 L26A 0.23 0.21 F27A 0.58 0.36 L30A 0.01
0.0032 D32A 0.64 0.62 R33A 0.0015 0.00022 H34A 0.71 0.4 D35A 0.78
0.3 Q40A 0.97 0.91 D114R 0.86 0.46 L117A 0.14 0.18 R120A 0.014
0.0005 R120E <0.0005 <0.0005 R125A 0.80 0.87 R125E 1.1 0.41
K131A 0.77 0.48 E132A 0.95 0.41 K133A 0.35 0.23 R144A 0.042 0.018
A145G 0.18 0.13 M148A 0.05 0.052 R149A 0.022 0.017 S152A 0.32 0.47
L153A 0.1 0.31 N156A 1.8 1.3 H57Y, E58N, Q61S, L30A 0.34 0.13 H57Y,
E58N, Q61S, R33A 0.073 0.0082 H57Y, E58N, Q61S, M148A 0.45 0.94
H57Y, E58N, Q61S, L153A 1.06 2.3 N65A, L80A, Y85A, Y89A 0.012
0.0009 N65A, L80A, Y85A, Y89A, D114A 0.019 0.0005 N65A, L80A, Y85A,
Y89A, L117A 0.0003 <0.0005 N65A, L80A, Y85A, Y89A, R120A
<0.00001 <0.00001 Y85A, Y89A, R120A 0.005 <0.0003 D114A,
R120A 0.017 0.002 L117A, R120A 0.0015 <0.0005 L117A, R120A,
K121A 0.003 <0.0005 R120A, K121A 0.031 <0.0009 R120E, K121E
<0.00002 <0.0002 .DELTA.(L161-E165) 0.72 1.1
TABLE-US-00008 TABLE 7 Relative affinity of interferon mutants to
their receptors Affinity to Affinity to IFNAR1 IFNAR2 IFN.alpha.2b
wild type 1 1 L15A 0.079 A19W 0.82 R22A 0.73 R23A 0.51 S25A 0.92
L26A 0.12 F27A 0.22 L30A 0.46 0.0015 L30V 0.0097 K31A 0.32 D32A
0.34 R33K 0.00031 R33A 0.57 0.000087 R33Q 0.000029 H34A 0.37 D35A
0.64 Q40A 0.91 D114R 2.5 L117A 0.45 0.77 R120A ND 0.71 R120E 1.4
R125A 1.1 R125E 1.1 K131A 0.46 E132A 1.5 K133A 0.11 K134A 0.75
R144A 0.027 A145G 0.03 A145M 0.15 M148A 0.02 R149A 0.0054 S152A
0.19 L153A 0.083 N156A 0.99 H57Y, E58N, Q61S, L30A 53 0.0011 H57Y,
E58N, Q61S, R33A 40 0.000069 H57Y, E58N, Q61S, M148A 43 0.22 H57Y,
E58N, Q61S, L153A 70 0.11 N65A, L80A, Y85A, Y89A ND 0.53 N65A,
L80A, Y85A, Y89A, D114A 1.1 N65A, L80A, Y85A, Y89A, L117A 1 N65A,
L80A, Y85A, Y89A, R120A ND Y85A, Y89A, R120A 0.91 D114A, R120A 0.83
L117A, R120A 1.4 L117A, R120A, K121A 0.14 0.91 R120A, K121A 1.7
R120E, K121E 1.3 .DELTA.(161-165) 0.53
[0194] These mutants have known reductions in binding to the type 1
interferon receptor IFNAR1 or IFNAR2, and/or have known reductions
in IFN.alpha. potency based on cell-based assays.
[0195] The data in these tables was disclosed in the following
references: [0196] Piehler, Jacob, Roisman, Laila C., Schreiber,
Gideon (2000). New structural and functional aspects of the Type I
interferon-receptor interaction revealed by comprehensive
mutational analysis of the binding interface. J. Biol. Chem. 275:
40425-40433. [0197] Jaitin, Diego A, Roisman, Laila C, Jaks, Eva,
Gavutis, Martynas, Piehler, Jacob, Van der Heyden, Jose, Uze,
Gilles, Schreiber, Gideon (2006). Inquiring into the differential
action of interferons (IFNs): an IFN-.alpha.2 mutant with enhanced
affinity to IFNAR1 is functionally similar to IFN-.beta.. Mol.
Cell. Biol. 26: 1888-1897. [0198] Slutzki, Michal, Jaitin, Diego
A., Yehezkel, Tuval Ben, Schreiber, Gideon (2006). Variations in
the unstructured C-terminal tail of interferons contribute to
differential receptor binding and biological activity. J. Mol.
Biol. 360: 1019-1030. [0199] Kalie, Eyal, Jaitin, Diego A.,
Abramovich, Renne, Schreiber, Gideon (2007). An interferon .alpha.2
mutant optimized by phage display for IFNAR1 binding confers
specifically enhanced antitubor activities. J. Biol. Chem. 282:
11602-11611. [0200] Pan, Manjing, Kalie, Eyal, Scaglione, Brian J.,
Raveche, Elizabeth S., Schreiber, Gideon, Langer, Jerome A. (2008).
Mutation of to IFNAR-1 receptor binding site of human IFN-.alpha.2
generates Type I IFN competitive antagonists. Biochemistry 47:
12018-12027. [0201] Kalie, Eyal, Jaitin, Diego A., Podoplelova,
Yulia, Piehler, Jacob, Schreiber, Gideon (2008). The Stability of
the ternary interferon-receptor complex rather than the affinity to
the individual subunits dictates differential biological
activities. J. Biol. Chem. 283: 32925-32936.
[0202] The abbreviation "YNS" is sometimes used herein to represent
IFN.alpha. variants including the following mutation: H57Y, E58N
and Q61S.
[0203] The present invention also contemplates combinations of the
abovementioned mutations or deletions in IFN.alpha..
[0204] The invention also contemplates the combination of the
constructs of the present invention with other drugs and/or in
addition to other treatment regimens or modalities such as
radiation therapy or surgery. When the contructs of the present
invention are used in combination with known therapeutic agents the
combination may be administered either in sequence (either
continuously or broken up by periods of no treatment) or
concurrently or as an admixture. In the case of cancer, there are
numerous known anticancer agents that may be used in this context.
Treatment in combination is also contemplated to encompass the
treatment with either the construct of the invention followed by a
known treatment, or treatment with a known agent followed by
treatment with the construct of the invention, for example, as
maintenance therapy. For example, in the treatment of cancer it is
contemplated that the constructs of the present invention may be
administered in combination with an alkylating agent (such as
mechlorethamine, cyclophosphamide, chlorambucil,
ifosfamidecysplatin, or platinum-containing alkylating-like agents
such as cysplatin, carboplatin and oxaliplatin), an antimetabolite
(such as a purine or pyrimidine analogue or an antifolate agent,
such as azathioprine and mercaptopurine), an anthracycline (such as
Daunorubicin, Doxorubicin, Epirubicin Idarubicin, Valrubicin,
Mitoxantrone, or anthracycline analog), a plant alkaloid (such as a
vinca alkaloid or a taxane, such as Vincristine, Vinblastine,
Vinorelbine, Vindesine, paclitaxel or Dosetaxel), a topoisomerase
inhibitor (such as a type I or type II topoisomerase inhibitor), a
Podophyllotoxin (such as etoposide or teniposide), or a tyrosine
kinase inhibitor (such as imatinib mesylate, Nilotinib, or
Dasatinib).
[0205] In the case of the treatment of multiple myeloma, it is
contemplated that the constructs of the present invention may be
administered in combination with current therapies, such as
steroids such as dexamethasone, proteasome inhibitors (such as
bortezomib or carfilzomib), immunomodulatory drugs (such as
thalidomide, lenalidomide or pomalidomide), or induction
chemotherapy followed by autologous haematopoietic stem cell
transplantation, with or without other chemotherapeutic agents such
as Melphalan hydrochloride or the chemotherapeutic agents listed
above.
[0206] In the case of the treatment of Hodgkin's lymphoma, it is
contemplated that the constructs of the present invention may be
administered in combination with current therapeutic approaches,
such as ABVD (Adriamycin (doxorubicin), bleomycin, vinblastine, and
dacarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine,
vincristine, mechlorethamine, etoposide, prednisone), or BEACOPP
(doxorubicin, bleomycin, vincristine, cyclophosphamide,
procarbazine, etoposide, prednisone).
[0207] In the case of non-Hodgkin's lymphoma or other lymphomas, it
is contemplated that the constructs of the present invention may be
administered in combination current therapeutic approaches.
Examples of drugs approved for non-Hodgkin lymphoma include
Abitrexate (Methotrexate), Adriamycin PFS (Doxorubicin
Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride),
Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Arranon
(Nelarabine), Bendamustine Hydrochloride, Bexxar (Tositumomab and
Iodine I 131 Tositumomab), Blenoxane (Bleomycin), Bleomycin,
Bortezomib, Chlorambucil, Clafen (Cyclophosphamide),
Cyclophosphamide, Cytoxan (Cyclophosphamide), Denileukin Diftitox,
DepoCyt (Liposomal Cytarabine), Doxorubicin Hydrochloride,
DTIC-Dome (Dacarbazine), Folex (Methotrexate), Folex PFS
(Methotrexate), Folotyn (Pralatrexate), Ibritumomab Tiuxetan,
Istodax (Romidepsin), Leukeran (Chlorambucil), Linfolizin
(Chlorambucil), Liposomal Cytarabine, Matulane (Procarbazine
Hydrochloride), Methotrexate, Methotrexate LPF (Methotrexate),
Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mozobil
(Plerixafor), Nelarabine, Neosar (Cyclophosphamide), Ontak
(Denileukin Diftitox), Plerixafor, Pralatrexate, Rituxan
(Rituximab), Rituximab, Romidepsin, Tositumomab and Iodine I 131
Tositumomab, Treanda (Bendamustine Hydrochloride), Velban
(Vinblastine Sulfate), Velcade (Bortezomib), and Velsar
(Vinblastine Sulfate), Vinblastine Sulfate, Vincasar PFS
(Vincristine Sulfate), Vincristine Sulfate, Vorinostat, Zevalin
(Ibritumomab Tiuxetan), Zolinza (Vorinostat). Examples of drug
combinations used in treating non-Hodgkin lymphoma include CHOP
(C=Cyclophosphamide, H=Doxorubicin Hydrochloride
(Hydroxydaunomycin), O=Vincristine Sulfate (Oncovin),
P=Prednisone); COPP (C=Cyclophosphamide, O=Vincristine Sulfate
(Oncovin), P=Procarbazine Hydrochloride, P=Prednisone); CVP
(C=Cyclophosphamide, V=Vincristine Sulfate, P=Prednisone); EPOCH
(E=Etoposide, P=Prednisone, O=Vincristine Sulfate (Oncovin),
C=Cyclophosphamide, H=Doxorubicin Hydrochloride
(Hydroxydaunomycin)); ICE (I=Ifosfamide, C=Carboplatin,
E=Etoposide) and R-CHOP (R=Rituximab, C=Cyclophosphamide,
H=Doxorubicin Hydrochloride (Hydroxydaunomycin), O=Vincristine
Sulfate (Oncovin), P=Prednisone.
[0208] Combination of retinoids with interferon-based fusion
protein constructs is also contemplated. Retinoids are a family of
molecules that play a major role in many biological functions
including growth, vision, reproduction, epithelial cell
differentiation and immune function (Meyskens, F. et al. Crit Rev
Oncol Hematol 3:75, 1987, Herold, M. et al. Acta Dermatovener 74:29
1975). Early preclinical studies with the retinol all-trans
retinoic acid or ATRA, either alone or in combination with other
agents, demonstrated activity against acute promyelocytic leukemia
(APL), myelodysplastic syndrome, chronic myelogenous leukemia
(CML), mycosis fungoides and multiple myeloma (reviewed in Smith,
M. J. Clin. Oncol. 10:839, 1992). These studies led to the approval
of ATRA for the treatment of APL. Currently there are over 100
clinical trials evaluating the activity of ATRA in combination with
other therapies for the treatment of hematological malignancies,
kidney cancers, lung cancers, squamous cell carcinomas and more. Of
particular interest and pertaining directly to this invention are
the studies demonstrating enhanced efficacy of interferon-.alpha.
treatment when combined with ATRA. This is described for mantle
cell lymphoma (Col, J. et al. Cancer Res. 72:1825, 2012), renal
cell carcinoma (Aass, N. et al. J. Clin. Oncol. 23:4172, 2005;
Motzer, R. J. Clin. Oncol. 18:2972, 2000), CML, melanoma, myeloma
and renal cell carcinoma (Kast, R. Cancer Biology and Therapy,
7:1515, 2008) and breast cancer (Recchia, F. et al. J. Interferon
Cytokine Res. 15:605, 1995). We would therefor predict enhanced
activity of our targeted attenuated IFNs when combined with
therapeutic dosing of ATRA in the clinic. In addition, Mehta (Mol
Cancer Ther 3(3):345-52, 2004) demonstrated that in vitro treatment
of leukemia cells with retinoic acid induced expression of CD38
antigen. Thus, the enhanced efficacy of interferon plus the induced
expression of the target CD38 would indicate a combination therapy
of ATRA with our anti-CD38 antibody-attenuated IFN.alpha. in the
treatment of IFN-sensitive cancers that express CD38 or may be
induced by ATRA to express CD38. Example of such cancers are
multiple myeloma, non-Hodgekin's lymphoma, CML and AML.
[0209] In addition, while the above constructs are based on
IFN.alpha.2b, the mutations or deletions could also be made in the
context of any of the other IFN.alpha.s or IFN.beta.. In another
embodiment of the present invention, the type I IFN is an
IFN.beta.. IFN-.beta. is approved for the treatment of multiple
sclerosis (MS). IFN-.beta. could be attenuated by mutation or
deletion and then attached to an antibody that targets cells
involved in the pathogenesis of this disease. IFN-.beta. is an
effective drug in MS, but its use is associated with adverse
events, including injection site inflammation, flu-like symptoms,
leukocytopenia, liver dysfunction and depression, leading to
discontinuation in a subset of patients. By directing IFN-.beta.
activity directly to pathogenic cells, these adverse events may be
avoided.
[0210] Pathogenesis of MS is thought to be initiated and progressed
by a number of events, including innate activation of dendritic and
microglial cells through toll-like receptors, an imbalance between
pro-inflammatory and anti-inflammatory/regulatory cytokines,
differentiation of CD4+ T cells into Th1 and Th17 phenotypes,
activation of Th1 cells by antigen presenting cells (APCs),
reduction in the number of regulatory T (Treg) cells and migration
of activated immune cells across the blood-brain barrier (BBB). The
primary drivers of the clinical episodes of the disease are thought
to be autoreactive, myelin-specific Th1 cells (reviewed in Gandhi,
2010 J Neuroimmunol 221:7; Boppana, 2011 Mt Sinai J Med 78:207;
Loma, 2011 Curr Neuropharmacol 9:409).
[0211] In an embodiment of the invention, an attenuated version of
IFN-.beta. may be attached to an antibody targeting a cell surface
marker specific for T cells, for the treatment of multiple
sclerosis or other autoimmune indications where IFN-.beta. may be
effective. Direct effects of IFN-.beta. on T cells include
inhibition of proliferation (Rep, 1996 J Neuroimmunol 67:111),
downregulation of the co-stimulatory molecule CD40L (Teleshova,
2000 Scand J Immunol. 51:312), decrease of metaloproteinase
activity leading to reduced migration across the BBB (Stuve, 1996
Ann Neurol 40:853; Uhm, 1999 Ann Neurol 46:319), induction of
apoptosis by upregulating intracellular CTLA-4 and cell surface Fas
molecules (Hallal-Longo, 2007 J Interferon Cytokine Res 27:865),
downregulation of anti-apoptotic proteins (Sharief, 2001 J
Neuroimmunol. 120:199; Sharief, 2002 J Neuroimmunol. 129:224), and
restoration of Treg function (De Andres, 2007 J Neuroimmunol
182:204; Korporal, 2008 Arch Neurol 65:1434; Sarasella, 2008 FASEB
J 22:3500; Chen, 2012 J Neuroimmunol 242:39).
[0212] Therefore, in one aspect of the present invention, an
attenuated IFN-.beta. is attached to an anti-CD3 antibody that
targets all T cells, which includes CD4+, CD8+, Treg, Th1, Th2 and
Th17 cells. This comprehensive approach ensures full coverage of
all T cells, as all of these cell types have reported roles in MS
pathogenesis and are affected by IFN-.beta. treatment (Dhib-Jalbut,
2010 Neurology 74:S17; Prinz, 2010 Trends Mol Med 16:379; Graber,
2010 Clin Neurol Neurosurg 112:58 and Loma, 2011 Curr
Neuropharmacol 9:409). Examples of CD3 antibodies that may be
incorporated into the fusion protein constructs of the present
invention are listed in Table 8.
TABLE-US-00009 TABLE 8 CD3 Antibodies Ab Clones Patent Assignee
Comments TF NSO U.S. Pat. No. 7,994,289 BTG International Humanized
CD3A.122 M291 U.S. Pat. No. 7,381,803 PDL BioPharma Humanized 28F1,
27H5, U.S. Pat. No. 7,728,114 Novimmune S.A. Human 23F10, 15C3
[0213] Alternatively, an attenuated IFN-.beta.-anti-CD4 fusion
protein construct presents a more restrictive approach, but would
target autoreactive and regulatory T cells, including Th1 and Th17
cells and CD4.sup.+CD25.sup.+ Treg cells. In addition, subsets of
dendritic cells (DCs) also express CD4 and direct therapeutic
effects of IFN-.beta. on DCs have been disclosed (Shinohara, 2008
Immunity 29:68; Dann, 2012 Nat Neurosci 15:98). Examples of CD4
antibodies that may be incorporated into the fusion protein
constructs of the present invention are listed in Table 9.
TABLE-US-00010 TABLE 9 CD4 Antibodies Ab Clones Patent Assignee
Comments CE9.1 U.S. Pat. No. 7,452,534 Biogen Idec Non-human
primate variable regions TRX1 U.S. Pat. No. 7,541,443 Tolerrx
Humanized 1E11, 1G2, 6G5, U.S. Pat. No. 8,231,877 GenPharm Human
10C5, 4D1
[0214] A role for CD8.sup.+ T cells in MS has been reported
(Friese, 2005 Brain 128:1747; Friese, 2009 Ann Neurol 66:132), as
well as a direct effect of IFN-.beta. on CD8+ T cells in MS
patients (Zafranskaya, 2006 Immunol 121:29). Therefore, directing
an attenuated IFN-.beta. directly to CD8.sup.+ T cells with an
anti-CD8 antibody may result in clinical benefits for MS patients.
Examples of CD8 antibodies are shown in Table 10.
TABLE-US-00011 TABLE 10 CD8 Antibodies Ab Clones Patent Assignee
Comments 37B1, 8G6 U.S. Pat. No. Ortho-McNeil Hybridomas 7,247,474
Pharmaceutical, Inc. deposited at ATCC (HB-12441, HB- 12657) OKT8
U.S. Pat. No. Ortho Hybridoma 4,361,550 Pharmaceutical deposited at
ATCC Corporation (CRL-8014) Several US2009/ Baylor Research
examples 0304659 Institute
[0215] Markers of activated T cells, including, but not limited to
CD25, CD38, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, ICOS and
PD-1, also represent attractive targets for this approach, since
activated T cells are thought to be the main drivers of
autoreactivity resulting in demyelination in MS (Gandhi, 2010 J
Neuroimmunol 221:7; Boppana, 2011 Mt Sinai J Med 78:207; Loma, 2011
Curr Neuropharmacol 9:409). Antibodies targeting any of these
antigens could be attached to an attenuated IFN.beta.. Examples
antibodies that could be used in the present invention include the
following: CD71 antibodies include BA120g (U.S. Pat. No. 7,736,647)
and various antibodies mentioned in Wang et at (Di Yi Jun Yi Da Xue
Xue Bao (Academic journal of the first medical college of PLA)
22(5):409-411, 2002). Examples of antibodies to CD83 include 20B08,
6G05, 20D04, 11G05, 14C12, 96G08 and 95F04 (U.S. Pat. No.
7,700,740). An example of an antibody to CD86 includes 1G10H6D10
(U.S. Pat. No. 6,071,519). HLA-DR antibodies include HD3, HD4, HD6,
HD7, HD8 and HD10 (U.S. Pat. No. 7,262,278), DN1921 and DN1924
(US2005/0208048). One attractive target along these lines could be
PD-1, which is expressed on recently activated T cells. Ideally, a
non-antagonizing antibody could be used, such as the J110 antibody
discussed in further detail below.
[0216] Examples of antibodies to ICOS include JMabs (U.S. Pat. No.
6,803,039) and JMab 136 (US2011/0243929).
[0217] Further of these examples of antibodies to these targets are
shown in the Tables 11 and
TABLE-US-00012 TABLE 11 CD25 Antibodies Ab Clones Patent Assignee
Comments `Anti-tac` Abs U.S. Pat. No. PDL, Inc daclizumab 5,530,101
RFT5 U.S. Pat. No. Novartis AG Chimeric, inhibits 6,521,230 MLR
AB1, AB7, AB11, U.S. Pat. No. Genmab A/S Human antibodies, AB12
8,182,812 (or prevent CD25-IL-2 U.S. Pat. No. interaction and
7,438,907) inhibit MLR
TABLE-US-00013 TABLE 12 CD44 Antibodies Ab Clones Patent Assignee
Comments H90 US2007/0237761 Chimeric 1A9, 2D1, 14G9, US2010/0092484
Human 10C8 SACK-1 U.S. Pat. No. 7,816,500 Sackstein Binds CD44
glycoforms
[0218] In another embodiment of the invention, an attenuated
version of IFN-.beta. can be fused to an antibody targeting cell
surface markers of myeloid cells, known to contribute to MS
pathogenesis by driving T cell activation and differentiation. For
example, the pan-myeloid markers CD33, CD115, or the dendritic cell
marker CD11c may be targeted. A broad targeting approach may be
preferred, for example, using antibodies against CD33 or CD115,
since the exact contribution of each of the myeloid cell subsets to
MS disease pathogenesis and response to IFN-.beta. has been
disputed (Prinz, 2008 Immunity 28:675; Shinohara, 2008 Immunity
29:68; Dann, 2012 Nat Neurosci 15:98). Antibodies to CD33 that
could be used in the present invention include My9-6 (U.S. Pat. No.
7,557,189), any of 14 antibodies described in US patent application
US2012/0082670, or the antibody known as huM195 (U.S. Pat. No.
5,693,761). Antibodies to CD115 that could be used include Ab1 and
Ab16 (U.S. Pat. No. 8,206,715) or CXIIG6 (US2011/0178278). An
example of a CD11c antibody that could be used according to the
present invention is mab 107 (U.S. Pat. No. 7,998,738, ATCC deposit
number PTA-11614). The attenuated IFN-.beta. could alternatively be
directed to the CD14 antigen, present primarily on macrophages.
Examples of CD14 antibodies are shown in Table 13.
TABLE-US-00014 TABLE 13 CD14 Antibodies Ab Clones Patent Assignee
Comments 4C1 U.S. Pat. No. 6,245,897 Seikagaku Mouse Ab Corporation
F1024-1-3 U.S. Pat. No. 7,264,967 Mochida Humanized, Pharmaceutical
inhibits Co. CD14/TLR binding F1024, U.S. Pat. No. 8,252,905/
Mochida Part of fusion F1031-13-2 US Pharmaceutical proteins with
2008/0286290 Co. protease
[0219] In yet another embodiment, targeting CD52-expressing cells
would deliver IFN-.beta. to all lymphocytes and, in addition, to
monocytes and peripheral dendritic cells (Buggins, 2002 Blood
100:1715; Ratzinger, 2003 Blood 101:1422), which are the key APCs
responsible for proliferation and differentiation of autoreactive T
cells in MS. This approach would direct the activity of IFN-.beta.
to the key cell types known to be directly affected by IFN-.beta.
and would facilitate its therapeutic activity in MS. Examples of
CD52 antibodies that could be used according to the present
invention include, but are not limited to DIVHv5/DIVKv2 (U.S. Pat.
No. 7,910,104), any of the CD52 antibodies disclosed in
(US2012/0100152) or CAMPATH.
[0220] Any of the above mentioned, antibody-targeted attenuated
IFN.beta. fusion protein constructs may have therapeutic activity
in the context of other inflammatory and autoimmune diseases beyond
multiple sclerosis, due to their common underlying immunological
etiologies.
[0221] Autoimmune diseases contemplated herein include inter alia
alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease multiple sclerosis, autoimmune disease
of the adrenal gland, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune oophoritis and orchitis, Behcet's disease,
bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis,
chronic fatigue syndrome (CFIDS), chronic inflammatory
demyelinating, chronic inflammatory polyneuropathy, Churg-Strauss
syndrome, cicatricial pemphigoid, crest syndrome, cold agglutinin
disease, Crohn's disease, irritable bowel syndrome, inflammatory
bowel disease, dermatitis herpetiformis, discoid lupus, essential
mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's
disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
nephropathy, insulin dependent diabetes (Type I), lichen planus,
lupus, Meniere's disease, mixed connective tissue disease, multiple
sclerosis, myasthenia gravis, myocarditis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychondritis,
polyglancular syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, pochitis, primary agammaglobulinemia, primary
biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's
syndrome, rheumatic fever, rheumatoid arrthritis, sarcoidosis,
scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus
erythematosus, Takayasu arteritis, temporal arteritis/giant cell
arteritis, ulcerative colitis, uveitis, vasculitis and vitiligo. Of
particular interest is Behcet's disease and chronic uveitic macular
edema and other types of uveitis, since IFN.alpha. has been shown
to render therapeutic benefit (Deuter, Dev Ophthalmol. 51:90-7.
2012)
[0222] Examples of inflammatory disease conditions contemplated by
the present disclosure include but are not limited to those disease
and disorders which result in a response of redness, swelling,
pain, and a feeling of heat in certain areas that is meant to
protect tissues affected by injury or disease. Inflammatory
diseases which can be treated using the methods of the present
disclosure, include, without being limited to, acne, angina,
arthritis, aspiration pneumonia, disease, empyema, gastroenteritis,
inflammation, intestinal flu, NEC, necrotizing enterocolitis,
pelvic inflammatory disease, pharyngitis, PID, pleurisy, raw
throat, redness, rubor, sore throat, stomach flu and urinary tract
infections, chronic inflammatory demyelinating polyneuropathy,
chronic inflammatory demyelinating polyradiculoneuropathy, chronic
inflammatory demyelinating polyneuropathy, chronic inflammatory
demyelinating polyradiculoneuropathy.
[0223] The sequence of human interferon-.beta.1 is shown below:
(SEQ ID NO: 191)
TABLE-US-00015 * * * * 1 MSYNLLGFLQ RSSNFQCQKL LWQLNGRLEY
CLKDRMNFDI PEEIKQLQQF 50 * * * 51 QKEDAALTIY EMLQNIFAIF RQDSSSTGWN
ETIVENLLAN VYHQINHLKT 100 * ** * * 101 VLEEKLEKED FTRGKLMSSL
HLKRYYGRIL HYLKAKEYSH CAWTIVRVEI 150 * 151 LRNFYFINRL TGYLRN
166
[0224] Using the numbering scheme above (residues 1-166), known
mutations (at positions indicated by asterisks) in human IFN.beta.
that reduce its activity include those listed in Table 14.
TABLE-US-00016 TABLE 14 IFN.beta. activity-attenuating mutations
IFNbeta Fold mutations attenuation* Reference wild type 1 R27A 3.3
1 R35A + C17S 280 3 R35T 10 1 E42K >10 2 D54N 1.4 2 M62I 8.7 2
G78S 6.2 2 K123 2.5 1 C141Y >25 2 A142T >10 2 R147A + C17S**
1.7 3 E149K >5 2 R152H 4.7 2 *based on anti-proliferation
activity **the C17S mutation was made in order to remove the
unpaired cysteine in the native sequence of IFN.beta.1
REFERENCES
[0225] (1) Runkel, L., Pfeffer, L., Lewerenz, M., Mogensen, K.
(1998). Differences in Activity between .alpha. and .beta. Type I
Interferons Explored by Mutational Analysis. J. Biol. Chem. 273:
8003-8008 [0226] (2) Stewart, A. G., Adair, J. R., Catlin, G.,
Hynes, C., Hall, J., Dav ies, J., Dawson, K. & Porter, A. G.
(1987). Chemical mutagenesis of human interferon-beta:
construction, expression in E. coli, and biological activity of
sodium bisulfite-induced mutations. DNA 6: 119-128. [0227] (3)
In-house results
[0228] In still another embodiment of the present invention, the
IFN is IFN-.lamda. (WO 2007/029041 A2), which may be used for any
of the applications described more thoroughly for IFN.alpha., or
IFN|3.
[0229] Type I IFNs can have anti-cancer activity based on a direct
stimulation of the type I IFN receptor on cancer cells. This has
been shown for numerous types of cancer including multiple myeloma,
melanoma, B cell lymphoma, non-small cell lung cancer, renal cell
carcinoma, hairy cell leukemia, chronic myelogenous leukemia,
ovarian cancer, fibrosarcoma, cervical cancer, bladder cancer,
astrocytoma, pancreatic cancer, etc (Borden, Cancer Research
42:4948-53, 1982; Chawla-Sarkar, Clinical Cancer Research 7:
1821-31, 2001; Morgensen, Int J. Cancer 28:575-82, 1981; Otsuka,
British Journal of Haematology 103:518-529, 1998; Lindner, J of
Interferon and Cytokine Research 17:681-693, 1997; Caraglia, Cell
Death and Differentiation 6:773-80, 1999; Ma, World J Gastroenterol
11(10):1521-8, 2005). One of skill in the art will recognize that
the present invention has many aspects resulting from combining
antibodies to tumor associated antigens with mutated type I
interferons, and that the resulting fusion protein constructs may
be used to reduce the proliferation of various interferon-sensitive
cancers that express the corresponding tumor associated antigens.
It will also be appreciated that type I interferons can be combined
with other agents to further improve their effectiveness.
[0230] Type I interferons can also display anti-viral properties.
IFN.alpha.2b, for example, has been FDA-approved for the treatment
of chronic hepatitis C infections, and may have utility in treating
other viral infections as well. Pegylated IFN-.alpha. is currently
part of the standard of care regimen for hepatitis C, according to
American and European guidelines, but results in side effects in
over 80% of patients, often leading to discontinuation of treatment
(Aman, 2012; Calvaruso, 2011). In one aspect of the present
invention, a type I IFN with an attenuating mutation is attached to
an antibody that binds to virally infected cells. The antigen to be
recognized by the above referenced antibody could be a viral
protein that is transiently expressed on the host cell surface, or
it could be an endogenous host cell-produced antigen that is
exposed on the cell surface to a greater extent after viral
infection than before infection. Exemplary viral proteins that
could serve as targets for the antibody portion include but are not
limited to, Hepatitis C viral envelope glycoproteins, E1 and E2;
Hepatitis B surface antigen (HBsAg); Herpes virus viral envelope
glycoproteins B, C, D, E, G, H, I, J, K, L, M and UL32, and
envelope protein UL49A; Human immune deficiency virus (HIV)
Envelope proteins glycoprotein (gp) 120 and gp41; Adenoviruses knob
domain of the fiber protein; Varicella-zoster virus envelope
glycoproteins (gB, gC, gE, gH, gI, gK, gL); Epstein-barr virus
viral glycoprotein gp350 and viral protein BMRF-2; Human
cytomegalovirus UL16; Parvovirus B19 viral capsid proteins VP1-3;
Human astrovirus structural proteins, e.g. VP26, VP29 and VP32;
Noroviruses structural protein VP1 and capsid protein VP2;
Poliovirus viral capsid proteins VP0, VP1, VP2, VP3 and VP4;
Rhinovirus viral capsid proteins VP1, VP2, VP3 and VP4; and dengue
virus virus particle proteins capsid (C), pre-membrane/membrane
(prM/M) and envelope (E).
[0231] In one embodiment, IFN-.alpha. activity may be targeted with
an antibody that binds, directly or indirectly via an intermediate
protein such as annexin V or beta2-glycoprotein 1, to
phosphatidylserine (PS), a phospholipid component of the inner
leaflet of cellular membranes. Cells undergoing apoptosis, however,
or cells infected with viruses, expose PS on the outer membrane,
where it becomes accessible to antibodies. PS is exposed on the
surface of cancer cells (Reidl, L. et al., J Immunol. 14:3623,
1991), the vascular endothelium in tumors (Ran, S. et al., Cancer
Res. 62:6132. 2002; He, J. et al., Clin Cancer Res. 15:6871, 2009),
and virus-infected cells (Soares, M. et al., Nat Med. 14:1357,
2008). An antibody indirectly (via beta2 glycoprotein 1) targeting
PS, bavituximab, has been described. It mediates antibody-dependent
cytotoxicity and is effective in a number of in vivo cancer models,
including human breast and lymphoma xenografts and a rat
glioblastoma model, as well as in viral disease models (Ran, S. et
al., Clin Cancer Res; 11:1551, 2005; He, J. et al., Clin Cancer
Res. 15:6871, 2009; Soares, M. et al., Nat Med. 14:1357, 2008).
Currently, it is being developed as a therapeutic antibody for lung
cancer treatment (DeRose, P. et al., Immunotherapy. 3:933, 2011;
Gerber, D. et al., Clin Cancer Res. 17:6888, 2011). Alternative
antibodies may be based on the variable regions from the anti-PS
antibody 9D2 (Cancer Res Nov. 1, 2002 62; 6132). Yet another
alternative for targeting PS would be to replace the antibody Fab
portions with a natural PS-binding protein such annexin V or
beta2-glycoprotein 1. An anti-PS antibody (or alternatively a
direct or indirect PS binding protein) fused with an attenuated
version of IFN-.alpha. would target IFN-.alpha. activity to PS
expressing virus-infected cells without displaying the systemic
safety issues related to IFN-.alpha.. Certain tumor cells, such as
lung cancer cells, also express PS on their cell surfaces, so an
antibody (or alternatively a direct or indirect PS binding protein)
to PS, attached to an attenuated IFN, could also have use in the
treatment of certain cancers.
[0232] It should be understood that antibody-targeted attenuated
IFN, could also be used in much the same way as IFN.alpha. for the
targeting virally infected cells (S. V. Kotenko, G. Gallagher, V.
V. Baurin et al., "IFN-.lamda.s mediate antiviral protection
through a distinct class II cytokine receptor complex," Nature
Immunology, vol. 4, no. 1, pp. 69-77, 2003).
[0233] In one embodiment Type II IFNs, namely INFy, may also be
attenuated and attached to antibodies that direct them to specific
cell types. IFN.gamma. has anti-proliferative properties towards
cancer cells (Kalvakolanu, Histol. Histopathol 15:523-37, 2000; Xu,
Cancer Research 58:2832-7, 1998; Chawla-Sarkar, Apoptosis 8:237-49,
2003; Schiller, J Interferon Resarch 6:615-25, 1986). Sharifi has
described how to make a fusion protein in which an IFN.gamma. has
been fused to the C-terminus of a tumor-targeting antibody
(Sharifi, Hybridoma and Hybridomics 21(6):421-32, 2002). In this
reference, Sharifi disclosed how to produce antibody-IFN.gamma.
fusion proteins in mammalian cells and showed that both the
antibody and the IFN were functional. Alternatively, a single-chain
dimer version of IFN.gamma., as described by Lander (J Mol Biol.
2000 May 26; 299(1):169-79) may be used in the fusion protein. In
addition to IFN.gamma.'s anti-proliferative effect on the targeted
tumor cells, it may also have another effect specifically on breast
cancer cells: IFN.gamma. has been shown to restore antiestrogen
sensitivity to breast cancer cells (Mol Cancer Ther. 2010 May;
9(5): 1274-1285) and so an attenuated-IFN.gamma. attached to a
breast cancer antigen antibody may be therapeutically useful in
combination with antiestrogen therapy. By attenuating IFN.gamma.
via mutation, a more cancer-selective form of IFN.gamma. may be
produced. Two attenuating mutations in IFN.gamma. have been
described by Waschutza (Eur J. Biochem. 256:303-9, 1998), namely
des-(A23, D24), in which residues A23 and D24 are deleted, and
des-(N25, G26), in which residues N25 and G26 are deleted. The
des-(A23, D24) mutant has an .about.18-fold reduced affinity for
the IFN.gamma. receptor compared to wild type IFN.gamma., and had a
.about.100-fold reduced antiviral activity compared to wild type
IFN.gamma.. The des-(N25, G26) variant had a .about.140-fold
reduced affinity for the IFN.gamma. receptor compared to wild type
IFN.gamma., and had a .about.10-fold reduced antiviral activity
compared to wild type IFN.gamma.. Examples of fusion proteins
comprising antibodies to tumor cell surface targets and attenuated
mutants of IFN.gamma. include the following: Rituximab may be used
as fusion protein with one of these attenuated IFN.gamma. using a 7
amino acid linker described by Sharifi to produce the fusion
protein construct "Rituximab-HC-L7-IFN.gamma.(.DELTA.[A23,D24])
IgG1," composed of SEQ ID NOS:378 (heavy chain) and 276 (light
chain)). Such a fusion protein construct would be expected to have
potent anti-proliferative activity against CD20 malignancies such
as B cell lymphomas. Other attenuated mutants of IFN.gamma. that
may be appropriate for fusing to a cell-targeting antibody were
described by Lundell (J Biol. Chem. 269(23):16159-62, 1994), namely
S20I (.about.50.times. reduced affinity), D21K (.about.100.times.
reduced affinity), A23Q (.about.2,500-fold reduced binding), A23V
(.about.2,000-fold reduced binding) and D24A (.about.4-fold reduced
binding). These attenuated IFN.gamma. may be used as fusions in
combination with anti-CD38 antibodies, to generate the fusion
protein construct "X355/02-HC-L7-IFN.gamma.(S20I) IgG1" (composed
of SEQID NOS:380 (heavy chain) and 226 (light chain)) or
"R10A2-HC-L7-IFN.gamma.(D21K) IgG1" (composed of SEQ ID NOS:382
(heavy chain) and 270 (light chain)). Other attenuating mutations
in IFN.gamma. that may be exploited for the current invention were
described by Fish (Drug Des Deliv. 1988 February;
2(3):191-206.)
[0234] Targeted attenuated IFN.gamma. may also be used to treat
various indications characterized by pathological fibrosis,
including kidney fibrosis, liver fibrosis and idiopathic pulmonary
fibrosis (IPF). IPF is a chronic, progressive form of lung disease,
characterized by fibrosis of unknown cause, occurring primarily in
older adults. Despite the medical need, there has been little
progress in the development of effective therapeutic strategies
(O'Connell, 2011 Adv Ther 28:986). Pulmonary fibrosis can also be
induced by exposure to drugs, particles, microorganisms or
irradiation. The following relates to both IPF and lung fibrosis
induced by known agents and potentially for treatment of fibrosis
in other types of organs, including liver and kidney.
[0235] Fibroblasts play a key role in fibrotic diseases of the lung
and their activation leads to collagen disposition, resulting in
excessive scarring and destruction of the lung architecture. Yet
there is little information on the origin of these pathogenic
fibroblasts, though several precursor cell types have been
proposed, including bone marrow progenitors, monocytes, circulating
fibrocytes, and endogeneous cells, such as resident mesenchymal and
epithelial cells (Stevens, 2008 Proc Am Thorac Soc 5:783; King,
2011 Lancet 378:1949).
[0236] CD14.sup.+ monocytes from peripheral blood are able to
differentiate into fibrocytes, the precursors of fibroblasts, and
this process is inhibited by interferon-.gamma. (IFN-.gamma.). A
direct effect of IFN-.gamma. on monocytes was demonstrated in in
vitro differentiation studies, supporting the strategy of targeting
an attenuated form of IFN-.gamma. to CD14.sup.+ monocytes for the
treatment of fibrotic disease (Shao, 2008 J Leukoc Biol
83:1323).
[0237] Experimental evidence exists that IFN-.gamma. is capable of
inhibiting proliferation and activation of fibroblasts (Rogliani,
2008 Ther Adv Respir Dis 2:75) and this fact has exploited
successfully in preclinical models to reduce scaring and fibrosis.
Clinical trials in IPF patients studying the benefit of
subcutaneously administered IFN-.gamma. failed to reach primary
endpoints for survival benefits (O'Connell, 2011 Adv Ther 28:986;
King, 2011). Current approaches focus on direct delivery of
recombinant IFN-.gamma. through inhalation of an aerosol form
(Diaz, 2012 J Aerosol Med Pulm Drug Deliv 25:79), such that the
lungs may achieve sufficient IFN-.gamma. activity to produce
benefit at an overall safe systemic dose.
[0238] Delivering IFN-.gamma. activity directly to fibroblasts
could be a powerful method to increase clinical response to this
agent and at the same time reduce its side effects. Fusing
attenuated IFN-.gamma. to antibodies targeting fibroblast specific
markers could facilitate this approach. There are several
fibroblast cell surface molecules that are enriched in fibroblasts.
These include, for example, fibroblast specific protein (FSP1;
Strutz, 1995 J Cell Biol 130:393), fibroblast activation protein
(FAP; Park, 1999 J Biol Chem 274:36505; Acharya, 2006 Hum Pathol
37:352), and platelet derived growth factor receptors
(PDGFR-.alpha. and -.beta.; Trojanowska, 2008 Rheumatology (Oxford)
47S5:2). Expression of these molecules is elevated in lung biopsies
obtained from IPF patients and they have been directly implicated
as drug targets in IPF or its pathogenesis (Lawson, 2005 Am J
Respir Crit Care Med 171:899; Acharya, 2006 Hum Pathol 37:352;
Abdollahi, 2005 J Exp Med 201:925). Examples of antibodies to FAP
and the PDGF receptors are shown in Tables 15 and 16.
TABLE-US-00017 TABLE 15 FAP Antibodies Ab Clones Patent Assignee
Comments MFP5, BIBH1 US2009/0304718 Boehringer Humanized Ingelheim
USA Corporation Many US2012/0128591 Bacac et al. Humanized F19
US2003/0143229 Boehringer Ingelheim International GmbH
TABLE-US-00018 TABLE 16 PDGFR-.alpha. and -.beta. Antibodies Ab
Clones Patent Assignee Comments 2.175.3, 2.499.1, U.S. Pat. No.
7,754,859 AstraZeneca AB Human abs against 2.998.2 PDGFR.alpha.
IMC-3G3 US2012/0027767 Imclone LLC Human abs against PDGFR.alpha.
2C5 US2012/00221267 Imclone LLC Human abs against PDGFR.beta.
[0239] In a preclinical model of liver fibrosis, IFN-.gamma. was
delivered to hepatic stellate cells, the equivalent of fibroblasts
and responsible for secreting collagen in liver fibrosis, through
liposomes targeting PDGFR-.beta., thereby enhancing the
anti-fibrotic effects of IFN-.gamma. (Li, 2012 J Control Release
159:261). These data support the concept and the potential
therapeutic benefit gained by delivering IFN-.gamma. activity
directly to fibroblasts in fibrotic diseases, including IPF and
liver fibrosis, and validate PDGFR-.beta. as a target for this
approach.
[0240] The present invention also contemplates the attenuation and
antibody-based targeting of type III IFNs, including IFN.lamda.1
(IL29), IFN.lamda.2 (IL28A), and IFN.lamda.3 (IL28B) (S. V.
Kotenko, G. Gallagher, V. V. Baurin et al., "IFN-.lamda.s mediate
antiviral protection through a distinct class II cytokine receptor
complex," Nature Immunology, vol. 4, no. 1, pp. 69-77, 2003., P.
Sheppard, W. Kindsvogel, W. Xu, et al., "IL-28, IL-29 and their
class II cytokine receptor IL-28R," Nature Immunology, vol. 4, no.
1, pp. 63-68, 2003). These IFNs act through receptors composed of
the IFNAR1 chain (also known as IL28R.alpha.) and the IL10R2 chain
(shared with IL10, IL22, and IL26 receptor complexes [A. Lasfar, W.
Abushahba, M. Balan, and K. A. Cohen-Solal, "Interferon lambda: a
new sword in cancer immunotherapy," Clinical and Developmental
Immunology, vol. 2011, Article ID 349575, 11 pages, 2011]). IFNARs
are expressed on most cell types and mediate similar signalling
pathways as the type I IFNs. The antiviral activity of .lamda. IFNs
has been demonstrated against several viruses including HBV and HCV
(E. M. Coccia, M. Severa, E. Giacomini et al., "Viral infection and
toll-like receptor agonists induce a differential expression of
type I and .lamda. interferons in humans plasmacytoid and
monocyte-derived dendritic cells," European Journal of Immunology,
vol. 34, no. 3, pp. 796-805, 2004; M. D. Robek, B. S. Boyd, and F.
V. Chisari, "Lambda interferon inhibits hepatitis B and C virus
replication," Journal of Virology, vol. 79, no. 6, pp. 3851-3854,
2005; N. Ank, H. West, C. Bartholdy, K. Eriksson, A. R. Thomsen,
and S. R. Paludan, "Lambda interferon (IFN-.lamda.), a type III
IFN, is induced by viruses and IFNs and displays potent antiviral
activity against select virus infections in vivo," Journal of
Virology, vol. 80, no. 9, pp. 4501-4509, 2006; S. E. Doyle, H.
Schreckhise, K. Khuu-Duong et al., "Interleukin-29 uses a type 1
interferon-like program to promote antiviral responses in human
hepatocytes," Journal of Hepatology, vol. 44, no. 4, pp. 896-906,
2006; T. Marcello, A. Grakoui, G. Barba-Spaeth et al., "Interferons
.alpha. and .lamda. inhibit hepatitis C virus replication with
distinct signal transduction and gene regulation kinetics,"
Gastroenterology, vol. 131, no. 6, pp. 1887-1898, 2006). Clinical
studies with IFN.lamda. for the treatment of hepatitis C have shown
promise (E. L. Ramos, "Preclinical and clinical development of
pegylated interferon-lambda 1 in chronic hepatitis C," Journal of
Interferon and Cytokine Research, vol. 30, no. 8, pp. 591-595,
2010). One aspect of the present invention is to target a mutated,
attenuated for of an IFN.lamda. towards virally infected cells,
using for example the targeting antibodies describe above for the
targeting of an attenuated form of IFN.alpha.. Mutated, attenuated
forms of an IFN.lamda. could also be used to target cancer cells,
as described in more detail for IFN.alpha., above.
[0241] Non-IFN ligands are also contemplated in the present
invention and may also be attenuated by mutation and then targeted
to specific cell types by antibodies or fragments thereof. The
anti-inflammatory cytokine interleukin-10 (IL-10) plays a central
role during innate and adaptive immune responses. IL-10 forms a
homodimer and binds to the IL-10 receptor complex expressed on
APCs, leading to reduced expression of MHC class II and reduced
production of pro-inflammatory cytokines and chemokines, thereby
inhibiting T cell development and differentiation. However, IL-10
has also been implicated in inducing the proliferation of several
immune cells, including B cells (Hofmann, 2012 Clin Immunol
143:116).
[0242] Reduced expression of IL-10 is associated with a number of
autoimmune disorders in humans and rodents, including psoriasis,
inflammatory bowel disease and rheumatoid arthritis. Mice deficient
in IL-10 develop chronic enterocolitis, which can be prevented by
the administration of IL-10, but the clinical translation of these
findings resulted in a number of failed trials in patients. One
explanation of these failures is that the local IL-10
concentrations may be too low, even at maximum tolerable systemic
administration (Herfarth, 2002 Gut 50:146). Another explanation may
be the immunostimulatory effect of IL-10 on B cells and the
resulting production of the pro-inflammatory IFN-.gamma., as was
demonstrated in IL-10-treated Crohn's disease patients (Tilg, 2002
Gut 50:191).
[0243] Fusing attenuated IL-10 to an antibody specific for APCs,
e.g. targeting dendritic cells through CD11c, or more broadly
expressed myeloid markers, like CD33 or CD115, would decrease
systemically active biologic activity and at the same time increase
the targeted local active concentrations of IL-10. In addition, the
demonstrated pro-inflammatory effect through B cells would be
decreased or eliminated. The production of antibody-IL10 fusion
proteins have been described previously (Schwager Arthritis Res
Ther. 11(5): R142, 2009).
[0244] Evidence exists for an anti-fibrotic role of IL-10 in
various models. A hallmark of fibrosis is the overproduction and
deposition of collagen produced by fibroblasts, resulting in
scarring tissue formation. IL-10 directly inhibits extracellular
matrix synthesis by human fibroblasts (Reitamo, 1994 J Clin Invest
94:2489) and is anti-fibrotic in a rat hepatic fibrosis model
through downregulation of TGF-.beta. (Shi, 2006 World J
Gastroenterol 12:2357; Zhang, 2007 Hepatogastroenterology 54:2092).
Clinical use of IL-10 is hampered by its short half-life and a
PEGylated version has shown promising pharmacokinetic improvements
and efficacy in a preclinical model of fibrosis (Mattos, 2012 J
Control Release 162:84). Targeting IL-10 activity through fusion
with an antibody directing it to fibroblasts could result in
therapeutic benefits in fibrotic diseases, including lung and liver
fibrosis. Antibodies against fibroblast specific proteins such as
fibroblast activation protein and platelet derived growth factor
receptors, as described above in the description of
IFN-.gamma.-targeting, could deliver attenuated IL-10 directly to
fibroblasts.
[0245] Recombinant erythropoietin (EPO) is a widely used and
effective hormone for the treatment of anemia, often in cancer
patients. It acts by signaling through the EPO receptor (EPOR),
which is not only expressed by cells of the hematopoietic system,
but also on non-hematopoietic cells, including cells from various
tumor types. Many studies have examined the role of EPO and EPO-R
stimulation in cancer models in vitro and in vivo, and a number of
studies have demonstrated a stimulatory effect on tumor growth,
either directly on cancer cells, or through increased angiogenesis
in the tumors (reviewed in Jelkmann, 2008 Crit Rev Oncol Hematol
67:39). In several clinical trials, treatment with EPO has been
associated with increased tumor growth and decreased survival,
leading to the recommendation and black box warning to limit and
monitor the exposure of EPO in cancer patients as much as
clinically feasible (Farrell, 2004 The Oncologist 9:18; Jelkmann,
2008 Crit Rev Oncol Hematol 67:39; Elliott, 2012).
[0246] Erythropoiesis is a multi-step process, in which pluripotent
stem cells undergo tightly controlled differentiation and
proliferation steps. An intermediate cell type in this process, is
the colony-forming-unit-erythroid (CFU-E) cell, which expresses
high levels of EPOR, depends on EPO for survival and appears to be
the main cell type in the differentiation process with this
dependency (Elliott, 2008 Exp Hematol 36:1573).
[0247] Targeting EPO activity to CFU-E cells using specific markers
would substantially reduce the effect of EPO on cancer and other
non-hematopoietic cells, while maintaining the ability to drive
erythrocyte formation and increase hemoglobin levels. Genome-wide
analysis of CFU-E cells revealed several potential candidate
cellular markers, including Rh-associated glycoproteins, e.g. CD241
and members of the Rh blood group system, e.g. the product of the
RCHE gene (Terszowski, 2005 Blood 105:1937).
[0248] Additional example surface markers expressed on CFU-Es, and
several other intermediates of erythropoiesis, include CD117
(c-kit), CD71 (transferrin receptor) and CD36 (thrombospondin
receptor) (Elliott, 2012 Biologics 6:163), but these markers are
overexpressed in certain cancer cells as well, as they are all
involved in general growth and proliferation, and therefore
represent less attractive targets for targeting EPO activity in
cancer patients, but this approach may benefit patients with tumors
not expressing these targets. CD117 antibodies include SR-1 (U.S.
Pat. No. 7,915,391) and antibodies DSM ACC 2007, 2008 and 2009
(U.S. Pat. No. 5,545,533). Other antigens for targeting of an
attenuated EPO include CD34, CD45RO, CD45RA, CD115, CD168, CD235,
CD236, CD237, CD238, CD239 and CD240.
[0249] Fusing EPO activity to an antibody would also greatly
increase the extent of the therapeutic activity. The half-life of
recombinant EPO is about 5 hours in humans and this would likely be
increased to weeks when attenuated EPO is fused to an antibody.
This approach could benefit patients treated for anemia, who are
dosed typically multiple times per week, often through intravenous
injections. Importantly, it has been shown that the therapeutic
response to EPO is primarily controlled by the length of time EPO
concentrations are maintained, and not by the concentration levels
(Elliott, 2008 Exp Hematol 36:1573).
[0250] Another example is transforming growth factor .beta.
(TGF-.beta.) which is a critical factor in the regulation of T
cell-mediated immune responses and the induction of immune
tolerance. TGF-.beta. knockout mice die from multifocal
inflammation and autoimmune disorders, suggesting an
immunosuppressive effect (Shull, 1992 Nature 359:693). However,
TGF-.beta. also has been shown to induce fibrotic disease through a
prominent role in extracellular matrix regulation and by promoting
fibroblast migration, proliferation and activation (Rosenbloom,
2010 Ann Intern Med 152:159; Wynn, 2011 J Exp Med 208:1339; King,
2011 Lancet 378:1949).
[0251] In the presence of TGF-.beta., CD4.sup.+CD25.sup.- naive T
cells can be converted into Treg cells, which can suppress
antigen-specific T cell expansion in vivo and prevent allergic
pathogenesis in a murine asthma model (Chen, 2003 J Exp Med
198:1875). Inflammatory responses also contribute to the transition
of acute liver disease and perpetuation into chronic fibrosis and
cirrhosis and TGF-.beta. may help dampen these responses through
its effect on Treg differentiation (Dooley, 2012 Cell Tissue Res
347:245). Similarly, TGF-.beta. directed to naive T cells in
inflammatory bowel disease could lead to control and suppression of
inflammation (Feagins, 2010 Inflamm Bowel Dis 16:1963).
[0252] Targeting TGF-.beta. specifically to CD4.sup.+ T cells may
leverage the anti-inflammatory potential of TGF-.beta., while
minimizing its pro-fibrotic properties, and could provide a novel
strategy to combat autoimmune disorders. Alternatively, TGF-.beta.
could be targeted soley to activated T cells using a T cell
activation marker, as described above for the discussion of
IFN.beta. targeting. One attractive target along these lines could
be, for example, PD-1, which is expressed on recently activated CD4
T cells. Ideally, a non-antagonizing antibody could be used, such
as the J110 antibody discussed in further detail below.
[0253] Another example is Interleukin-4 (IL-4) which is a cytokine
that induces the differentiation of naive CD4+ T cells into Th2
cells. Upon activation, Th2 cells produce more IL-4, and as a
result, IL-4 is considered a main driver of Th2-mediated immune
responses. The concept of a Th1/Th2 imbalance (favoring Th1)
contributing to autoimmune and other inflammatory diseases was
first postulated in the 1980s (reviewed in Kidd, 2003 Altern Med
Rev 8:223), and indeed, a role of Th1/Th17 cells as drivers of
disease in psoriasis (Ghoreschi, 2007 Clin Dermatol 25:574),
certain types of inflammatory bowel disease, in particular Crohn's
disease (Sanchez-Munoz, 2008 World J Gastroenterol 14:4280), or
severe versus mild forms of asthma (Hansbro, 2011 Br J Pharmacol
163:81), has been documented.
[0254] In preclinical models of infectious diseases, deviation of
the immune response away from Th1 to Th2 and activation of
macrophages by IL-4 protected from immunopathology (Hunig, 2010 Med
Microbiol Immunol 199:239), and IL-4 therapy of psoriasis patients
resulted in an induction of Th2 differentiation and an improvement
in clinical scores (Ghoreschi, 2003 Nat Med 9:40).
[0255] Diversion towards Th2 may provide a therapeutic benefit in
certain types of diseases. Delivery of IL-4 to CD4.sup.+ T cells
could accomplish this, or IL-4 activity could be targeted to
macrophages to protect from immunopathology (Ghoreschi, 2007 Clin
Dermatol 25:574; Hunig, 2010 Med Microbiol Immunol 199:239).
[0256] Attenuating mutations in IL-4 that may be exploited in the
design of antibody-attenuated IL-4 fusion protein constructs of the
present invention include those listed in Table 17.
TABLE-US-00019 TABLE 17 IL4 Variant K.sub.off .times. 10.sup.3
S.sup.-1 EC.sub.50 T cell proliferation (nM) IL4 2.1 0.12 I5R 8.7
T6D 15 E9Q 270 3.1 R81E 6.1 K84D 9.3 R88Q 140 2.5 R88A 760 8.1 N89R
6.1 W91D 8.5 ND. No specific binding found.
[0257] The IL-4 mutants in this table, and their binding properties
and biological activity, were described by Wang Y, Shen B and
Sebald W. Proc. Natl. Acad. Sci. USA 1997 March 4; 94(5):
1657-62.
[0258] In yet another example, Interleukin-6 (IL-6) may also be
attenuated and targeted to specific cell types. A mechanism by
which tumors can evade anti-tumor immunity is by recruiting Treg
cells to the tumor microenvironment, resulting in tolerance at
tumor sites. IL-6 is a cytokine involved in regulating the balance
between Treg and Th17 cells and induces the development of Th17
cells, while it inhibits Treg differentiation (Kimura, 2010 Eur J
Immunol 40:1830).
[0259] IL-6, by skewing the terminal differentiation of naive
CD4.sup.+ T cells towards the Th17 lineage, or reprogramming of
Th17 cells, has the potential to reverse tumor-associated immune
suppression by Treg cells in the context of cancer, thereby
enabling the immune system to control the tumors.
[0260] This strategy has proven successful in a murine model of
pancreatic cancer in which mice injected with tumor cells
expressing IL-6 demonstrated a significant delay in tumor growth
and enhanced survival, accompanied by an increase in Th17 cells in
the tumor microenvironment, compared to mice bearing tumors not
expressing IL-6 (Gnerlich, 2010 J Immunol 185:4063).
[0261] Adoptive transfer of T cells is an effective treatment for
solid (Rosenberg, 2011 Clin Cancer Res 17:4550) and hematologic
(Kochenderfer, 2012 Blood 119:2709) malignancies. Analysis of five
different clinical trials in which adoptive T cell transfer was
employed using a variety of preconditioning regimens revealed that
the depth and duration of Treg depletion correlates with clinical
response rate, highlighting the important role of residual Tregs
controlling the anti-tumor response (Yao, 2012 Blood 119:5688). In
mice, a direct link between surviving Tregs and efficacy of
adoptive transfer therapy strongly supports these clinical
observations (Baba, 2012 Blood 120:2417).
[0262] The importance of Tregs in controlling anti-tumor activity
is further exemplified by a significant increase in the humoral
response to peptide vaccination in glioblastoma patients after
depletion of Tregs with the anti-IL-2 receptor antibody daclizumab
(Sampson, 2012 PloS ONE 7:e31046).
[0263] Taken together, the published data strongly support a role
for Tregs in inhibiting the immune response against tumors. By
directing IL-6 activity to CD4.sup.+ cells in order to stimulate
Th17 differentiation and decrease Treg formation, enhanced
anti-tumor responses are expected. These may be achieved with or
without accompanying vaccination strategies. Fusing attenuated IL-6
to an antibody against a T cell antigen (e.g. targeting CD4) or an
activated T cell antigen (such as PD-1) would provide a
comprehensive delivery directly to the target cells.
[0264] Attenuated mutants of IL-6 include those listed in Table
18.
TABLE-US-00020 TABLE 18 EC.sub.50 in XG-1 growth stimulation assay
IL6 Variant Binding (% of wild type) (pg/ml) IL6 100 600 F74E 1 Low
activity F78E 5 Low activity R168M 2 Low activity R179E None
detected Low activity R179W None detected Low activity
[0265] These IL-6 mutants and their properties were described by
Kalai M et. al. Blood. 1997 Feb. 15; 89(4):1319-1333
[0266] Another example is hepatocyte growth factor (HGF) discovered
as a mitogen for hepatocytes (reviewed in Nakamura, 2010 Proc Jpn
Acad Ser B Phys Biol Sci 86:588). Hepatocyte growth factor is a
pleiotropic cytokine that regulates cell growth and motility,
playing a central role in angiogenesis and tissue generation and
repair in many organs.
[0267] HGF acts through its receptor, MET, which is expressed on
epithelial and endothelial cells. Binding of HGF to MET results in
a number of intracellular phosphorylation and signaling events,
leading to a variety of biological responses including migration,
proliferation and morphogenesis. Essential for embryogenesis, HGF's
primary function in the adult is tissue repair (Nakamura, 2010 Proc
Jpn Acad Ser B Phys Biol Sci 86:588).
[0268] HGF has been shown to alter the fate of epithelial cells and
reduce epithelial-mesenchymal transition (EMT) through its
intereference with TGF-.beta. signaling, antagonizing the process
of fibroblastogenesis (Shukla, 2009 Am J Respir Cell Mol Biol
40:643). After organ injury, TGF-.beta. drives conversion of
HGF-producing fibroblasts into collagen-producing myofibroblasts,
while HGF in turn inhibits TGF-.beta. production by myofibroblasts
(Mizuno, 2004 Am J Physiol Renal Physiol 286:F134). Exogeneous HGF,
or mimetics activating the MET receptor, act by restoring this
imbalance imposed by tissue injury, and are therefore considered
promising drug candidates for treating damaged tissues and fibrotic
diseases (Nakamura, 2010 Proc Jpn Acad Ser B Phys Biol Sci
86:588).
[0269] Initially studied in models for liver damage and hepatitis
(Roos, 1992 Endocrinology 131:2540; Ishiki, 1992 Hepatology
16:1227), HGF subsequently demonstrated therapeutic benefits in
many additional damaged organs, including pulmonary,
gastrointestinal, renal and cardiovascular models of injury and
fibrosis (Nakamura, 2011 J Gastroenterol Hepatol 26:188).
[0270] In in vivo model systems of fibrosis, HGF prevents the
progression of fibrotic changes and reduces collagen accumulation
when administered prophylactically or therapeutically in murine
lungs exposed to bleomycin (Yaekashiwa, 1997 Am J Respir Crit Care
Med 156:1937; Mizuno, 2005 FASEB J 19:580), in an obstructive
nephropathy model in mice (Yang, 2003 Am J Physiol Renal Physiol
284:F349) and in liver fibrosis models in rats (Matsuda, 1997
Hepatology 26:81); HGF also prevents fibrosis in cardiomyopathic
hamsters (Nakamura, 2005 Am J Physiol Heart Circ Physiol
288:H2131).
[0271] Limitations of HGF as a therapeutic include its short
half-life, which requires supra-physiological systemic
concentrations to reach locally effective levels, and the role of
its receptor, MET, in cancer. MET can activate oncogenic pathways
in epithelial cells. Both of these limitations may be overcome by
generation of an antibody-HGF fusion protein construct and
targeting it to regenerating or fibrotic tissue. This strategy
would produce a therapeutic with a much longer half-life directed
primarily at the relevant cells types.
[0272] Clinical trials have investigated the therapeutic potential
and regenerative activity of HGF, or HGF mimetics, in hepatic
failure, chronic leg ulcers, limb ischemia, peripheral arterial
disease, cardiovascular disease after myocardial infarction and
neurological diseases (de Andrade 2009 Curr Opin Rheumatol 21:649;
Nakamura, 2011 J Gastroenterol Hepatol 26:188; Madonna, 2012 Thromb
Haemost 107:656).
[0273] Liver fibrosis, typically the result of chronic liver damage
caused by infections or alcohol abuse, is, like fibrosis in other
organs, characterized by excessive accumulation of extracellular
matrix, including collagen produced by (myo) fibroblasts. Damaged
hepatocytes release inflammatory cytokines and the resulting
inflammatory milieu stimulates the transformation of hepatic
stellate cells (HSC) into fibroblasts, producing collagen. The
accumulation of extracellular matrix proteins results in scar
tissue, which leads to liver cirrhosis (Bataller, 2005 J Clin
Invest 115:209). Evidence exists for a direct effect of HGF on
hepatocytes and HSC in vitro (Kwiecinski, 2012 PloS One 6:e24568;
Namada, 2012 J Cell Physiol DOI 10.1002/jcp.24143). Targeting HGF
specifically to hepatocytes or HSC may result in a therapeutic
benefit in liver fibrosis patients, while eliminating the unwanted
systemic effects of HGF.
[0274] Possible membrane proteins for hepatocytes include, for
example, ASGR1, a subunit of the asialoglycoprotein, used as a
target for liver specific drug delivery (Stockert, 1995 Physiol Rev
75:591), or alternatively the other subunit of this receptor,
ASGR2. Fibroblast-specific protein (FSP1) expression is increased
after liver injury and may be used to target fibroblasts or
inflammatory macrophages in fibrotic liver tissue (Osterreicher,
2011 Proc Natl Acad Sci USA 108:308).
[0275] In lung fibrosis patients, the loss of pulmonary
architecture is characterized by a loss of alveolar epithelial
cells, the persistent proliferation of activated fibroblasts and
the extensive alteration of the extracellular matrix (Panganiban,
2011 Acta Pharmacol Sin 32:12).
[0276] To treat lung fibrosis, HGF activity may be delivered to
alveolar epithelial cells by attenuating it (by mutation) and
attaching it to an antibody against a specific cell surface protein
on these cells, such as RTI40/Tia or HTI56 (McElroy, 2004 Eur
Respir J 24:664).
[0277] Endothelial cell-specific markers, including VEGF receptors
(Stuttfeld, 2009 IUBMB Life 61:915) may be used for targeting blood
vessels for endothelial cell layer enhancement for a number of
pathologic indications, including hindlimb ischemia. Examples of
VEGF receptor antibodies are shown in Table 19.
TABLE-US-00021 TABLE 19 VEGFR Antibodies Ab Clones Patent Assignee
Comments AC88 U.S. Pat. No. 8,128,932 Shanghai Aosaiersi Human
anti-VEGFR2 Biotech Co., Ltd mAb Antibody 1, US2012/0058126 Imclone
LLC Anti-VEGFR3 Abs Antibody 2 6A6 US2011/0065176 Korea Research
Human, anti-VEGFR Institute of BioScience and BioTechnology
[0278] Many other examples of signaling ligands are also known in
the art and may, as described in the non-limiting exemplary
embodiments above, be attenuated and attached to an antibody (or
fragment thereof) that binds to an antigen on specific target
cells, thereby allowing the ligand to generate its biological
signal on those target cells to a much greater degree than it
generates its signal on antigen-negative cells. Examples of ligands
that have a direct negative effect on tumor proliferation include
TNF.alpha., TRAIL, Fas Ligand, IFN.beta., IFN.gamma. or IFN.lamda.,
which can be targeted to various tumor cell surface antigens as
discussed above for INF.alpha..
[0279] In many of the aspects of the present invention, specific
mutations in various ligands are explicitly mentioned. There are,
however, methods well known in the art for identifying other
mutations in signalling ligands numerous methods for mutagenesis of
proteins are known in the art. Such methods include random
mutagenesis for example, exposing the protein to UV radiation or
mutagenic chemicals and selecting mutants with desired
characteristics. Random mutagenesis may also be done by using doped
nucleotides in oligonucleotides synthesis, or conducting a PCR
reaction in conditions that enhance misincorporation of nucleotide,
thereby generating mutants. Another technique is site-directed
mutagenesis which introduces specific changes to the DNA. One
example of site directed mutagenesis is using mutagenic
oligonucleotides in a primer extension reaction with DNA
polymerase. This method allows for point mutation, or deletion or
insertion of small stretches of DNA to be introduced at specific
sites. The site-directed approach may be done systematically in
such technique as alanine scanning mutagenesis whereby residues are
systematically mutated to alanine and its effect on the peptide's
activity is determined. Each of the amino acid residues of the
peptide is analyzed in this manner to determine the important
regions of the peptide.
[0280] Another example is combinatorial mutagenesis which allows
the screening of a large number of mutants for a particular
characteristic. In this technique, a few selected positions or a
short stretch of DNA may be exhaustively modified to obtain a
comprehensive library of mutant proteins. One approach of this
technique is to excise a portion of DNA and replaced with a library
of sequences containing all possible combinations at the desired
mutation sites. The segment may be at an enzyme active site, or
sequences that have structural significance or immunogenic
property. A segment however may also be inserted randomly into the
gene in order to assess the structural or functional significance
of particular part of protein.
[0281] Methods of screening mutated ligands to determine potency
includes assaying for the presence of a complex between the ligand
and the target. One form of assay involves competitive binding
assays. In such competitive binding assays, the target is typically
labeled. Free target is separated from any putative complex and the
amount of free (i.e. uncomplexed) label is a measure of the binding
of the agent being tested to target molecule. One may also measure
the amount of bound, rather than free, target. It is also possible
to label the compound rather than the target and to measure the
amount of compound binding to target in the presence and in the
absence of the drug being tested.
[0282] One example of a cell free assay is a binding assay. Whilst
not directly addressing function, the ability of a modulator to
bind to a target molecule in a specific fashion is strong evidence
of a related biological effect. For example, binding of a molecule
to a target may, in and of itself, be inhibitory, due to steric,
allosteric or charge-charge interactions. The target may be either
free in solution, fixed to a support, expressed in or on the
surface of a cell. Either the target or the compound may be
labeled, thereby permitting determination of binding. Usually, the
target will be the labeled species, decreasing the chance that the
labeling will interfere with or enhance binding. Competitive
binding formats can be performed in which one of the agents is
labeled, and one may measure the amount of free label versus bound
label to determine the effect on binding.
[0283] Depending on the assay, culture may be required. The cell is
examined using any of a number of different physiologic assays.
Alternatively, molecular analysis may be performed, for example,
protein expression, mRNA expression (including differential display
of whole cell or polyA RNA) and others. Non-limiting examples of in
vitro biological assays that can be used to screen protein variants
are shown in the Examples below and also include apoptosis assays,
migration assays, invasion assays, caspase-activation assays,
cytokine production assays and the like.
[0284] The present invention also provides compositions comprising
the polypeptides of the present invention. These compositions can
further comprise at least one of any suitable auxiliary, such as,
but not limited to, diluent, binder, stabiliser, buffers, salts,
lipophilic solvents, preservative, adjuvant or the like.
Pharmaceutically acceptable auxiliaries are preferred. Non-limiting
examples of, and methods of preparing such sterile solutions are
well known in the art, such as, but not limited to, Gennaro, Ed.,
Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing
Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers can be
routinely selected that are suitable for the mode of
administration, solubility and/or stability of the antibody
composition as well known in the art or as described herein.
[0285] Pharmaceutical excipients and additives useful in the
present composition include but are not limited to proteins,
peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides;
derivatised sugars such as alditols, aldonic acids, esterified
sugars and the like; and polysaccharides or sugar polymers), which
can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein
excipients include serum albumin, such as human serum albumin
(HSA), recombinant human albumin (rHA), gelatin, casein, and the
like. Representative amino acids which can also function in a
buffering capacity include alanine, glycine, arginine, betaine,
histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,
isoleucine, valine, methionine, phenylalanine, aspartame, and the
like. One preferred amino acid is histidine. A second preferred
amino acid is arginine.
[0286] Carbohydrate excipients suitable for use in the invention
include, for example, monosaccharides, such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol
(glucitol), myoinositol and the like. Preferred carbohydrate
excipients for use in the present invention are mannitol,
trehalose, and raffinose.
[0287] Antibody compositions can also include a buffer or a pH
adjusting agent; typically, the buffer is a salt prepared from an
organic acid or base. Representative buffers include organic acid
salts, such as salts of citric acid, ascorbic acid, gluconic acid,
carbonic acid, tartaric acid, succinic acid, acetic acid, or
phthalic acid; Tris, tromethamine hydrochloride, or phosphate
buffers. Preferred buffers for use in the present compositions are
organic acid salts, such as citrate.
[0288] Additionally, the compositions of the invention can include
polymeric excipients/additives, such as polyvinylpyrrolidones,
ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such
as 2-hydroxypropyl-.beta.-cyclodextrin), polyethylene glycols,
flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, surfactants (e.g., polysorbates such as
"TWEEN.RTM. 20" and "TWEEN.RTM. 80"), lipids (e.g., phospholipids,
fatty acids), steroids (e.g., cholesterol), and chelating agents
(e.g., EDTA).
[0289] These and additional known pharmaceutical excipients and/or
additives suitable for use in the antibody compositions according
to the invention are known in the art, e.g., as listed in
"Remington: The Science & Practice of Pharmacy", 19 th ed.,
Williams & Williams, (1995), and in the "Physician's Desk
Reference", 52 nd ed., Medical Economics, Montvale, N.J. (1998),
the disclosures of which are entirely incorporated herein by
reference. Preferred carrier or excipient materials are
carbohydrates (e.g., saccharides and alditols) and buffers (e.g.,
citrate) or polymeric agents.
[0290] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0291] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an
admission that any or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present invention as it existed in Australia or elsewhere
before the priority date of each claim of this application.
[0292] It must be noted that, as used in the subject specification,
the singular forms "a", "an" and "the" include plural aspects
unless the context clearly dictates otherwise. Thus, for example,
reference to "a" includes a single as well as two or more;
reference to "an" includes a single as well as two or more;
reference to "the" includes a single as well as two or more and so
forth.
[0293] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
Examples of the Invention
Production of Antibody-IFN.alpha. Fusion Protein Constructs
Expression Vectors:
[0294] The DNA encoding the rituximab (Anderson et al., U.S. Pat.
No. 5,843,439, Dec. 1, 1998) and palivizumab (Johnson, U.S. Pat.
No. 5,824,307, Oct. 20, 1998) variable regions were generated from
18 (heavy chain) and 16 (light chain) DNA oligonucleotides, which
were designed according to the published amino acid sequences, by
PCR-based gene assembly. The DNA encoding the variable regions of
the G005 anti-CD38 and nBT062 anti-CD138 monoclonal antibodies were
drawn from the publications by De Weers et al. (U.S. Pat. No.
7,829,673) and by Daelken et al. (WO 2009/080832), respectively,
and subjected to be synthesized by Integrated DNA Technology, Inc.
(Coralville, Iowa) after the sequence modification to eliminate
rare codons and unprefered restriction sites.
[0295] The DNA sequences encoding the variable regions of
anti-human HLA (HB95), anti-human PD-1 (J110) and anti-yellow fever
virus (2D12) monoclonal antibodies were determined after cloning
from hybridoma W6/32 (ATCC HB-95, Barnstable et al. (1978), Cell
14:9-20), J110 (International Patent Organism Depositary FERM-8392,
Iwai et al. (2002), Immunol. Lett, 83:215-220) and 2D12 (ATCC
CRL-1689, Schlesinger et al. (1983), Virol. 125:8-17),
respectively, using the SMART RACE cDNA Amplification kit
(Clontech, Mountain View, Calif.) and Mouse Ig-Primer Sets
(Novagen/EMD Chemicals, San Diego, Calif.). The sequence
determination and sub-cloning of the newly isolated anti-CD38
antibodies is described in the following sections.
[0296] The DNA encoding human interferon-.alpha.2b (IFN.alpha.2b;
amino acid sequence of SEQ ID NO:3) was isolated from genomic DNA
of a HEK cell line by PCR. The sequences of human interferon-|31
(IFN.beta.1, SEQ ID NO:91), human interleukin-4 (IL-4, SEQ ID
NO:119) and human interleukin-6 (IL-6, SEQ ID NO:123) were designed
from the protein sequences such as NP_002167, NP_000580 and
NP_000591, respectively, and synthesized by Integrated DNA
Technology, Inc. (Coralville, Iowa) or GenScript USA Inc.
(Piscataway, N.J.) using methods commonly known to those of skill
in the art. Alterations of the cytokine sequences, for example the
addition of linkers or point mutations, were introduced to the
cytokine genes using overlap extension PCR techniques well known in
the art.
[0297] The cytokine-endoding gene fragments were then cloned into
the pTT5 expression vector (Durocher, Nucleic Acids Research volume
30, number 2, pages E1-9, 2002) containing either a human IgG1
heavy chain complete or partial constant region (such as Swissprot
accession number P01857), a human IgG4 heavy chain constant region
(such as Swissprot accession number P01861 incorporating
substitution S228P), human Ig kappa constant region (Swissprot
accession number P01834) or human Ig lambda constant region
(Swissprot accession number POCG05) either as a naked Ig or as a
cytokine gene fusion form using overlap extension PCR techniques
and restriction sites according to cloning methods well known by
those skilled in the art.
Production of IgG and IgG Interferon Fusion Protein Constructs:
[0298] DNA plasmids encoding the IgGs and IgG-cytokine fusion
protein constructs were prepared using Plasmid Plus Maxi kit
(Qiagen, Valencia, Calif.) and then transfected into HEK293-6E
cells (CNRC, Montreal, Canada) grown in F17 synthetic medium
supplemented with 0.1% Pluronic F-68, 4 mM L-glutamine (Invitrogen,
Carlsbad, Calif.) using a commercially available transfection
reagent and OptiMEM medium (Invitrogen, Carlsbad, Calif.). After
allowing for expression for 6 days in an incubator supplied with 5%
CO.sub.2 and gentle shaking, the culture media was isolated and
subjected to IgG affinity purification using Protein G-agarose
beads (GE Healthcare, Piscataway, N.J.). Purified IgG and
IgG-cytokine fusion protein constructs were then concentrated and
buffer-exchanged to phosphate buffered saline (PBS) pH 7.4 using
Amicon Ultra centrifugal filter devices (Millipore, Billerica,
Mass.), followed by protein concentration determination using a
NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham,
Mass.).
[0299] Although different antibody-cytokine fusion protein
constructs were expressed in the HEK system with differing yields,
several of them, in particular several of those based on
IFN.alpha., were produce at at least 100 mg/L of media, showed high
solubility and did not aggregate as determined by size exclusion
chromatography.
[0300] The amino acid sequences of the antibodies and
antibody-ligand construct fusion protein constructs are described
below. For antibody-cytokine fusion protein constructs in which the
cytokine was fused to the C-terminus of the heavy or light chain,
the following naming convention was used:
[name of mab]-[linkage to heavy chain ("HC") or light chain
("LC")]-[Linker name]-[ligand name] [(mutation)] [isotype].
[0301] Thus for example the construct "Rituximab-HC-L6-IFN.alpha.
(A145G) IgG1" is the antibody rituximab, with IFN.alpha.2b (with
the A145G point mutation), linked to the C-terminus of the IgG1
heavy chain, with an intervening linker L6.
[0302] The linkers used in the experiments were as follows:
L0: no linker (direct fusion of the C-terminus of an antibody chain
with the N-terminus of the cytokine)
TABLE-US-00022 (SEQ ID NO: 132) L6: SGGGGS (SEQ ID NO: 133) L16:
SGGGGSGGGGSGGGGS
Method for Measuring Antigen-Targeted Activity of
Antibody-IFN.alpha. Fusion Protein Constructs
[0303] "On target (Daudi) assay": This assay was used to quantify
the anti-proliferative activity of IFNs and antibody-IFN fusion
protein constructs on cells that display that antigen corresponding
to the antibody to which the IFN is fused, and may be used as part
of the assay for calculating the antigen-sensitivity index (ASI)
defined herein. Daudi cells express both CD20 and CD38 as cell
surface associated antigens. The viability of cells was measured
using the reagent CellTiter-Glo.RTM., Cat #G7570, from Promega
(Madison, Wis.). This is a luminescence-based assay that determines
the viability of cells in culture based on quantitation of ATP. The
signal strength is proportional to the number of viable cells in a
microtiter plate well. The details of the assay are as follows:
[0304] Daudi cells (obtained from ATCC, Manassas, Va.) were
cultured in a T75 flask (TPP, Trasadingen, Switzerland, cat#90076)
to a preferred density of between 0.5.times.10.sup.5 and
0.8.times.10.sup.5 viable cells/ml in RPMI 1640 (Mediatech, Inc.,
Manassas, Va., cat #10-040-CV) with 10% Fetal Bovine Serum (FBS;
Hyclone, Logan, Utah cat# SH30070.03). Cells were harvested by
centrifuging at 400 g for five minutes, decanting the supernatant,
and resuspending the cell pellet in RPMI 1640+10% FBS. Cells were
then counted andthe density was adjusted to 3.0.times.10.sup.5
cells/ml in RPMI 1640+10% FBS. Then, 50 .mu.l of the cell
suspension was aliquoted into each well of a 96 well round bottom
tissue culture plate (hereafter, "experimental plate") (TPP,
cat#92067). On a separate, sterile 96 well plate (hereafter,
"dilution plate"; Costar, Corning, N.Y. cat#3879), test articles
were serially diluted in duplicate in RPMI 1640+10% FBS. Then, 50
.mu.l/well was transferred from the dilution plate to the
experimental plate. The experimental plate was then incubated for
four days at 37.degree. C. with 5% CO.sub.2.
[0305] A mixture of the manufacturer-supplied assay buffer and
assay substrate (hereafter, "CellTiterGlo reagent", mixed according
to the manufacturer's instructions) was added to the experimental
plate at 100 The plate was shaken for two minutes. Then, 100
.mu.l/well was transferred from the experimental plate to a 96 well
flat bottom white opaque plate (hereafter, "assay plate"; BD
Biosciences, Franklin Lakes, N.J. cat#35 3296). The content of the
assay plate was then allowed to stabilize in the dark for 15
minutes at room temperature. The plate was read on a Victor 3V
Multilabel Counter (Perkin Elmer, Waltham, Mass., model#1420-041)
on the luminometry channel and the luminescence was measured.
Results are presented as "relative luminescence units (RLU)".
[0306] Data was analyzed using Prism 5 (Graphpad, San Diego,
Calif.) using non-linear regression and three parameter curve fit
to determine the midpoint of the curve (EC50). For each test
article, potency relative to free IFN.alpha.2b (or some other form
of IFN with a known potency relative to IFN.alpha.2b) was
calculated as a ratio of EC50s.
[0307] One of ordinary skill in the art will appreciate that there
are many other commonly used assays for measuring cell viability
that could also be used.
[0308] "On target (ARP) assay" (also sometimes referred to herein
as a "targeted assay"): The multiple myeloma cell line ARP-1 was a
gift from Bart Barlogie MD, PhD, Director of the Myeloma Institute
at the University of Arkansas Medical Center (Little Rock, Ak.). It
is described in Hardin J. et al., (Interleukin-6 prevents
dexamethasone-induced myeloma cell death. Blood; 84:3063, 1994).
ARP-1 cells (CD38.sup.+) were used to test CD38 targeting
antibody-IFN fusion protein constructs. Culture and assay
conditions were the same as for Daudi-based assay outlined above,
with the following exceptions: ARP-1 was cultured to a density of
4.0.times.10.sup.5 to 6.0.times.10.sup.5 cells/ml. ARP-1
concentration was adjusted to 1.0.times.10.sup.4 cells/ml prior to
assay.
Method for Measuring Non-Antigen-Targeted Activity of
Antibody-IFN.alpha. Fusion Protein Constructs
[0309] "Off-target assay" (also sometimes referred to herein as the
"not-targeted" assay): The iLite assay from PBL Interferon Source
(Piscataway, N.J., Cat#51100), was performed largely as described
by the manufacturer with the addition of a human IgG blocking step.
The iLite cell line is described by the manufacturer as "a stable
transfected cell line derived from a commercially available
pro-monocytic human cell line characterized by the expression of
MHC Class II antigens, in particular the human lymphocyte antigen
(HLA-DR), on the cell surface." The cell line contains a stably
transfected luciferase gene, the expression of which is driven by
an interferon-response element (IRE), which allows for interferon
activity to be quantified based on luminescence output. The
manufacturer-supplied iLite plate (hereafter "assay plate") and
diluent were removed from the -80.degree. C. freezer and allowed to
equilibrate to room temperature. Then, 50 .mu.l of the diluent was
added per well to the assay plate. The vial of
manufacturer-supplied reporter cells was removed from the
-80.degree. C. freezer and thawed in a 37.degree. C. water bath.
Then, 25 .mu.l aliquiots of cells were dispensed into each well of
the assay plate. Next, 12.5 .mu.l of 8 mg/ml human IgG that was
diluted into RPMI 1640+10% FBS (Sigma Chemicals, St. Louis, Mo.;
cat#14506) was added per well. The contents were mixed and
incubated at 37.degree. C. for 15 minutes. On a separate "dilution
plate," test articles were serially diluted in duplicate in RPMI
1640+10% FBS. Then, 12.5 .mu.l of the test articles were
transferred from the dilution plate to the assay plate. The assay
plate was then incubated at 37.degree. C. with 5% CO.sub.2 for 17
hours. The manufacturer-supplied assay buffer and substrate were
removed from the -80.degree. C. freezer and allowed to equilibrate
to room temperature for two hours. The manufacturer-supplied assay
buffer was added to the manufacturer-supplied substrate vial and
mixed well according to the manufacturer's instructions to create
the "luminescence solution." Then, 100 .mu.l of the luminescence
solution was added to each well of the assay plate. The plate was
shaken for 2 minutes. The plate was then incubated at room
temperature for 5 minutes in the dark and finally read on a Victor
3V Multilabel Counter on a luminometry channel and the luminescence
measured and presented as RLU. The data was analyzed with Graphpad
Prism 5 as described for the"on-target (Daudi) assay," above. To
test anti-CD38 antibody-IFN fusion protein constructs in the iLte
assay, the manufacturer-supplied diluent was supplenmented with 2
mg/ml human IgG and 0.5 mg/ml anti-CD38 antibody (same antibody
clone being tested as an antibody-IFN fusion protein construct, to
block any binding of the anti-CD38 antibody-IFN fusion protein
constructs to the CD38 expressed on the iLite cells).
Results: Antigen-Specificity of Antibody-IFN.alpha. Fusion Protein
Constructs
[0310] FIG. 6 shows the interferon activity of free IFN.alpha.2b
(SEQ ID NO:3; "IFN.alpha." in figure) as well as IFN.alpha.2b fused
to the C-terminus of the heavy chain of two different antibodies
(rituximab and palivizumab, an isotype control antibody), as acting
on a the iLite cell line. This cell line does not display the
antigen for either of these antibodies, so this assay reveals the
potency of various IFN.alpha.2b-containing proteins in the absence
of antibody-antigen-based targeting. The details of this assay are
described above under the heading "Method for measuring
non-antigen-targeted activity of antibody-IFN.alpha. fusion protein
constructs" and is hereafter abbreviated as the "off-target assay."
"Rituximab-HC-L6-IFN.alpha. IgG1" refers to the CD20-targeting
chimeric antibody Rituximab, in which the light chain (SEQ ID
NO:276) is unaltered but the IgG1 class heavy chain (SEQ ID NO:277)
has, attached to its C terminus, a 6 amino acid linker sequence
("L6;" SGGGGS, SEQ ID NO:132), followed by the sequence for
IFN.alpha.2b (SEQ ID NO:3); this heavy chain-linker-IFN.alpha.
sequence is shown as SEQ ID NO:280. "Isotype-HC-L6-IFN.alpha. IgG1"
refers to the RSV-targeting humanized antibody Palivizumab, in
which the light chain (SEQ ID NO:290) is unaltered but the IgG1
class heavy chain (SEQ ID NO:291) has, attached to its C terminus,
a 6 amino acid linker sequence ("L6;" SGGGGS, SEQ ID NO:132),
followed by the sequence for IFN.alpha.2b (SEQ ID NO:3); this heavy
chain-linker-IFN.alpha.2b sequence is shown as SEQ ID NO:294. In
this assay, free IFN.alpha.2b showed an EC.sub.50 for activating
gene expression through an interferon response element (IRE) of 1.9
pM. By attaching IFN.alpha.2b to Rituximab, there was a 3.1-fold
(5.9/1.9=3.1) decrease in its potency. A similar, modest decrease
in potency was observed when IFN.alpha.2b was linked to
Palivizumab. Again, the cell line used in this study did not have
the antigen corresponding to either of these antibodies on its cell
surface, demonstrating that attachment of an IgG to the N-terminus
of IFN.alpha.2b caused a modest (3-4.times.) decrease in its
non-antigen-targeted IFN activity. This is consistent with what has
been reported by other (for example in U.S. Pat. No. 7,456,257).
Neither Palivizumab nor Rituximab alone (without the fusion to an
interferon) showed any activity in this assay (data not shown).
[0311] To determine whether the antibody-IFN.alpha.2b fusion
protein constructs had enhanced activity relative to free
IFN.alpha.2b on cells that do display the corresponding antigen on
their cell surface, their effect on Daudi cells, which display the
CD20 antigen of Rituximab, but which do not display the RSV F
protein antigen corresponding to Palivizumab, was examined. The
assay used in this case, described above as "Method for measuring
antigen-targeted activity of antibody-IFN.alpha. fusion protein
constructs" or simply the "on-target (Daudi) assay," measured the
effect of the test substances on the viability of Daudi cells. With
these cells, the Rituximab-IFN.alpha.2b fusion protein construct
(Rituximab-HC-L6-IFN.alpha. IgG1) was 3.25-fold (1.3/0.4=3.25) more
potent than free IFN.alpha.2b (FIG. 7). In other words, the
attachment of Rituximab to IFN.alpha.2b resulted in slightly
reduced (3.1-fold) activity towards antigen-negative cells (FIG. 6)
but slightly increased (3.25-fold) activity towards
antigen-positive cells (FIG. 7). Overall, the antibody attachment
therefore increased the antigen-specificity index (ASI), defined as
the fold increased potency relative to free IFN.alpha.2b on
antigen-positive cells multiplied by the fold decreased potency
relative to free IFN.alpha.2b on antigen-negative cells, by 10-fold
(3.1.times.3.25) in this experiment. A repeat of the experiments
measured an ASI of 14, as shown in Table 20, row 2. The EC50
(mathematical midpoint of the dose-response curve) was used as a
measure of potency in the calculations presented here. In other
words, when compound A showed an EC50 that is 10-fold lower than
compound B, it was said to have a 10-fold higher potency.
[0312] The results presented in FIG. 8 are consistent with
antibody-based targeting relying on antibody-antigen reactivity:
the Rituximab-IFN.alpha. fusion protein construct
(Rituximab-HC-L6-IFN.alpha.-IgG1) was 12-fold (2.2/0.18=12) more
potent in reducing viability of the CD20.sup.+ Daudi cells than the
Palivizumab-IFN.alpha. fusion protein construct
(Isotype-HC-L6-IFN.alpha.-IgG1), the antigen for which is not
present on the Daudi cells.
[0313] The modest reduction in IFN.alpha. activity that occurred as
a result of linking it to an antibody may not be sufficient to
prevent the toxicity of the IFN.alpha. component of the construct
in human subjects. Various mutations were therefore introduced into
IFN.alpha.2b in order to reduce its activity and toxicity. For
example, five different mutant versions of IFN.alpha.2b were
generated and, in each case, linked to the C-terminus of the heavy
chain of Rituximab via the six amino acid linker L6, which has the
sequence SGGGGS (SEQ ID NO:132). These constructs were compared to
the the Rituximab-wild type IFN fusion protein construct,
Rituximab-HC-L6-IFN.alpha. IgG1 (as also used in the experiments
shown in FIGS. 6-8). The five mutant versions were R144A, A145G,
R33A+YNS, R33A and R144A+YNS. The sequences of these variants are
described below. The degree of expected reduced affinity for the
type I interferon receptors based on previous characterization by
others of IFN mutants, and the amount of expected attenuation in
interferon activity, are shown in Tables 6 and 7, above.
[0314] FIGS. 9, 10 and Table 20 show the degree of reduced
interferon activity for each of these Rituximab-attenuated
IFN.alpha.2b fusion protein constructs relative to free, wild type
IFN.alpha.2b, on antigen-negative (i.e. CD20-negative) cells. The
R144A mutant of the Rituximab-IFN.alpha.2b fusion protein construct
(composed of SEQ ID NOS:282 (heavy chain) and 276 (light chain))
showed 386-fold reduced interferon activity (2200/5.7=386). The
A145G and R33A+YNS versions (composed of the heavy chains of SEQ ID
NOS:284 and 286, respectively, each of which are combined with the
light chain of SEQ ID NO:276) showed 491-fold (2800/5.7=491) and
1,071-fold (6100/5.7=1,071) reduced activity, respectively. FIG. 10
shows the degree of reduced interferon activity for the R144A+YNS
fusion protein construct (composed of SEQ ID NOS:288 (heavy chain)
and 276 (light chain)) to be 303-fold (1700/5.6=303) relative to
the Rituxumab fusion protein construct lacking the IFN mutations
(Rituximab-HC-L6-IFN.alpha. IgG1); since Rituximab-HC-L6-IFN.alpha.
IgG1 is 3.8-fold less potent on antigen negative cells than free,
wild type IFN.alpha.2b (data from FIG. 9; 22/5.7=3.8), this means
that the R144A+YNS version of the fusion protein construct was
1,150-fold less potent than free, wild type IFN.alpha.
(303.times.3.8=1,150). The R33A version of the fusion protein
construct (composed of SEQ ID NOS:436 (heavy chain) and 276 (light
chain)) was attenuated to such a high degree that it showed no
detectable activity in the non-targeted assay.
TABLE-US-00023 TABLE 20 Targeted Non-Targeted Potency Relative
Potency Relative to free IFN.alpha.2b to free IFN.alpha.2b Antigen-
(EC50 IFN.alpha.2b/ (EC50 IFN.alpha.2b/ Specificity EC50 Fusion
EC50 Fusion Index (ASI; Fusion protein protein protein calculated
construct Test construct) construct) as Column A/ Article Column A
Column B Column B) Ritux-IFN.alpha.2b 3.6 0.26 14
Ritux-IFN.alpha.2b 0.86 0.0026 330 (R144A) Ritux-IFN.alpha.2b 1.2
0.0020 600 (A145G) Ritux-IFN.alpha.2b 1.6 0.00093 1,700 (R33A +
YNS) Ritux-IFN.alpha.2b 0.0022* No ND (R33A) detectable activity in
non-targeted assay Ritux-IFN.alpha.2b 0.23* 0.00086* 270 (R144A +
YNS) *Free IFN.alpha.2b was not tested on the same day as the test
articles in these rows. Therefore, these measurements are based on
a comparison of the test article with Rituximab-HC-L6-IFN.alpha.
IgG1, which was assayed on the same day and same plate, multiplied
by a correction factor based on the relative activity of
IFN.alpha.2b vs Rituximab-HC-L6-IFN.alpha. IgG1 (i.e. data shown in
the second row from the top) measured on a different day.
[0315] Surprisingly, when the amount of interferon activity of
these highly attenuated rituxumab-mutant IFN.alpha.2b fusion
protein constructs was measured on antigen-positive cells (Daudi,
CD20.sup.+), there was generally very little attenuation compared
to the wild type IFN.alpha.2b version of the Rituximab-IFN.alpha.2b
fusion protein construct (FIGS. 11-12), and thus the mutated
interferons still possessed the ability to activate the IFN
receptor on "on-target" cells whilst having a greatly reduced
ability to activate it on "off-target" cells. For example, the
R33A+YNS version of the construct was only 2.2-fold (0.74/0.33=2.2)
less active than the Rituximab-IFN.alpha.2b wild type construct on
the antigen-positive (Daudi) cells. This was in contrast to the
277-fold (6100/22=277; FIG. 9) reduced activity on antigen-negative
cells. The mutations in the IFN.alpha.2b, in the context of the
Rituximab-IFN.alpha.2b fusion protein construct, caused a
substantially greater attenuation of activity on antigen-negative
cells than on antigen-positive cells. As a result, the
Rituximab-HC-L6-IFN.alpha.2b (R33A+YNS) IgG1 fusion protein
construct exhibited a substantially greater antigen-specificity
index (ASI, 1,700-fold) compared to Rituximab-HC-L6-IFN.alpha.2b
IgG1 (10- to 14-fold) or free IFN.alpha.2b (1-fold, by definition),
suggesting that its off-target effects in vivo will be
substantially reduced.
[0316] Other Rituximab-IFN.alpha.2b constructs with mutations in
the IFN.alpha.2b portion also showed surprisingly little reduced
activity on antigen-positive cells (FIGS. 11 and 12) relative to
their reduced potency on antigen-negative cells (FIGS. 9 and 10).
With the exception of the R33A version of the fusion protein
construct, discussed below, the attenuating mutations caused a
384-1,160-fold decrease in interferon activity relative to free
wild type IFN.alpha.2b on antigen-negative cells, but showed
0.23-1.2-fold of the potency of wild type IFN.alpha.2b on
antigen-positive cells. The R33A mutated fusion protein construct,
which had undetectable IFN activity in the absence of
antibody-antigen targeting, still showed significant activity in
the presence of antibody-targeting; the potency of the R33A version
of the fusion protein construct was 1,620-fold lower than the same
fusion protein construct lacking this attenuating mutation in the
on-target assay (340/0.21=1,620-fold attenuation). This is in stark
contrast to the at least 100,000-fold attenuation caused by the
same mutation in the absence of antibody-based targeting (FIG. 10).
These results are summarized in Table 20.
[0317] To determine whether this dramatic difference in the ability
of the mutations in the IFN.alpha. component of the fusion protein
constructs to substantially reduce its activity on antigen-negative
cells as compared to antigen-positive cells could be extended to
other fusion protein constructs targeting other antigens,
antibodies targeting the multiple myeloma antigen CD 38 (SEQ ID
NO:131) were fused to both wild type and attenuated forms of
IFN.alpha. and characterized. Some of these experiments were
performed using the antibody G005 (De Weers et al. (U.S. Pat. No.
7,829,673)); the sequences of the heavy and light chains for this
human antibody are shown as SEQ ID NOS:135 and 134,
respectively.
[0318] In addition, several novel human and rat antibodies against
CD38 were produced, as described below.
Development of Novel CD38 Antibodies
Formatting CD38 Constructs for Expression
[0319] The extracellular domains (ECD) of human and cynomolgus
monkey CD38 proteins were each formatted to include a cleavable
N-terminal leader sequence, an Avitag.TM., a poly-histidine tag and
a thrombin cleavage site to yield proteins SEQID NO:127 and 128
respectively. These were back-translated into DNA sequences and
synthesized de novo by assembly of synthetic oligonucleotides by
methods known by those with skill in the art. Following gene
synthesis, the genes were subcloned into vector pTT5 (Durocher,
Nucleic Acids Research volume 30, number 2, pages E1-9, 2002) to
yield constructs to produce soluble secreted forms of these
proteins via transient expression in HEK293E cells (Durocher,
supra).
Construction of Vectors for Antibody Expression
[0320] Heavy and light chain variable region sequences were
subcloned into variants of the vector pTT5 containing either a
human IgG1 heavy chain constant region (such as Swissprot accession
number PO1857), a human IgG4 heavy chain constant region (such as
Swissprot accession number P01861 incorporating substitution
S228P), human kappa constant region (Swissprot accession number
P01834) or human lambda region (Swissprot accession number P0CG05)
to yield full length antibody chains.
Transient Expression of Constructs in HEK293-6E Cells
[0321] HEK293-6E cells were cultured in complete cell growth media
(1 L of F17 medium (Invitrogen.TM.), 9 mL of Pluronic F68
(Invitrogen.TM.), 2 mM Glutamine containing 20% (w/v) Tryptone NI
(Organotechnie.RTM.) with Geneticin (50 mg/mL, Invitrogen.TM.) at
50 .mu.l/100 mL culture). The day before transfection, cells were
harvested by centrifugation and resuspended in fresh media (without
Geneticin). The next day DNA was mixed with a commercial
transfection reagent and the DNA transfection mix added to the
culture drop-wise. The culture was incubated overnight at
37.degree. C. with 5% CO.sub.2 and 120 rpm without Geneticin. The
next day, 12.5 mL of Tryptone was added along with 250 .mu.l of
Geneticin per 500 mL culture. The culture was incubated at
37.degree. C., 5% CO.sub.2 and 120 rpm. After 7 days, the
supernatant was harvested by centrifugation and was ready for
purification.
Expression and Purification of Antibodies
[0322] Transient co-expression of heavy and light chains in
HEK293-6E cells (as described above) generated antibodies that were
subsequently purified by protein A chromatography. Briefly,
supernatants derived from these transfections were adjusted to pH
7.4 before being loaded onto a HiTrap Protein A column (5 mL, GE
Healthcare). The column was washed with 50 mL of 1.times.PBS (pH
7.4). Elution was performed using 0.1 M citric acid pH 2.5. The
eluted antibody was desalted using Zeba Desalting columns (Pierce)
into 1.times.PBS (pH 7.4). The antibodies were analyzed using
SDS-PAGE. The concentration of the antibody was determined using
the BCA assay kit (Pierce).
Purification of Histidine-Tagged Proteins from Tissue Culture
Supernatants
[0323] Immobilized metal ion affinity chromatography (IMAC) was
used to purify human and cynomolgous monkey CD38 extracellular
domain (ED) proteins from tissue culture supernatants. Briefly,
protein supernatants were diluted in binding buffer (20 mM sodium
phosphate, 0.5 M NaCl, 30 mM imidazole, pH 7.4) before being loaded
onto a HisTrap.TM. FF column (1 mL, GE Healthcare). The column was
washed with 5 mL of binding buffer (pH 7.4) and elution was
performed using 20 mM sodium phosphate, 0.5 M NaCl, 500 mM
imidazole, pH 7.4. The eluted proteins were desalted and buffer
exchanged using Amicon Ultra-15 centrifugal filter unit with
Ultracel-10 membrane (Millipore) into 1.times.PBS (pH 7.4). The
absorbance at 280 nm (A.sub.280) of the protein was assessed using
a Nanodrop spectrophotometer and readings corrected using the
predicted extinction coefficients to determine protein
concentrations.
Biotinylation of Antigens for Phage Display
[0324] The Avitag.TM. motifs of human and cynomolgus monkey CD38
EDs were biotinylated according to manufacturer's directions
(Avidity LLC, Aurora, Colo.). Excess unconjugated biotin was
removed from the biotinylated proteins by desalting into
1.times.PBS using a 7 KD molecular weight cut off (MWCO) Zeba spin
column (Thermo Scientific, Logan, Utah) according to manufacturer's
instructions. Successful biotinylation of CD38 ED proteins was
confirmed using a combination of polyacrylamide gel electrophoresis
and Western blotting. Western blots were probed using
Streptavidin-HRP (BD Biosciences, San Diego, Calif.) and developed
using TMB (Sigma-Aldrich, St. Louis, Mo.). For each antigen,
monomeric biotinylated CD38 ED was detected.
Generation of Anti-CD38 Antibodies by Phage Display
[0325] FAbs that bind to both human and cynomolgus monkey CD38 EDs
were isolated from a naive phagemid library comprising
approximately 2.5.times.10.sup.11 individual human FAb fragments.
Methods of generating phage antibody fragment libraries are
discussed in "Phage display: A Practical Approach" (Eds. Clackson
and Lowman; Oxford University Press, Oxford, UK) and "Antibody
Phage Display Methods and Protocols" (Eds. O'Brien and Aitken;
Humana Press Inc, NJ 07512). Briefly, antibody heavy and light
chain variable regions were amplified based on RNA from donor
samples. Antibody heavy and light chain variable regions were then
inserted into phagemid vectors to generate a library of antibody
fragments fused to a phage coat protein. The antibody library used
herein was a high diversity naive phagemid library that expressed
antibody fragments in the Fab format.
[0326] Anti-CD38 FAbs were isolated from the phage display library
over the course of two panning `campaigns` (i.e. discrete phage
display experiments with different reagents or panning conditions).
The general protocol followed the method outlined by Marks et al.
(Marks, J. D. & Bradbury, A., 2004, Methods Mol Biol, 248,
161-76).
[0327] Each phage display campaign involved three rounds of
panning. For each round, .about.2.5.times.10.sup.12 phage particles
were blocked by mixing 1:1 with blocking buffer (4% skim milk in
PBS, pH 7.4) and incubating for 1 hr at room temperature. The
blocked phage library was then pre-depleted for any biotinylated
protein tag motif binders used in panning through incubation for 45
mins with 50-200 pmols of an irrelevant antigen containing an
identical biotinylated tag motif. Tag- and streptavidin-binders
were captured by adding an excess (75-300 .mu.L) of
streptavidin-coated Dynabeads (Invitrogen), which were blocked as
described for the library. The beads (including tag- and
streptavidin-binders attached to them) were immobilized using a
magnet and discarded.
[0328] Library panning was conducted by mixing the blocked and
pre-depleted library with 50-200 pmols of biotinylated recombinant
CD38 ED in a 2 mL microcentrifuge tube and rotating for 2 hrs at
room temperature. Then, 100 .mu.L of streptavidin-coupled Dynabeads
(Invitrogen, Carlsbad, Calif.) were added and the mixture was
incubated a further 15 minutes as described previously.
Non-specifically bound phage were removed using a series of washes.
Each wash involved pulling the bead complexes out of the solution
onto the tube wall using a magnetic rack, aspirating the
supernatant and then re-suspending the beads in fresh wash buffer.
This was repeated multiple times with either PBS wash buffer
(1.times.PBS with 0.5% skim milk) or PBS-T wash buffer (1.times.PBS
supplemented with 0.05% TWEEN-20 [Sigma-Aldrich, St. Louis, Mo.]
and 0.5% skim milk). Phage that remained bound after the washing
process were eluted from the biotinylated-CD38 ED-bead complexes by
incubation with either a twenty-fold excess of non-biotinylated
CD38 ED for 1 hr at room temperature or 0.5 mL of 100 mM
triethylamine (TEA) (Merck Chemicals, Darmstadt) for 20 mins at
room temperature. TEA-eluted `output` phage were neutralized by the
addition of 0.25 mL of 1 M Tris-HCl pH 7.4 (Sigma-Aldrich, St.
Louis, Mo.).
[0329] At the end of the first and second rounds of panning, the
output phage were added to a 10 mL culture of exponentially growing
TG1 E. coli (2.times. yeast-tryptone (2YT) growth media) and
allowed to infect the cells during a 30 minute incubation at
37.degree. C. without shaking, then with shaking at 250 rpm for 30
additional minutes. The phagemids encoding the phage display output
were then rescued as phage particles following a standard protocol
(Marks, J. D. & Bradbury, A., 2004, Methods Mol Biol, 248,
161-76). At the end of the third panning round, TG1 cells were
infected with output phage and were plated on 2YT agar
(supplemented with 2% glucose and 100 .mu.g/mL carbenicillin) at a
sufficient dilution to produce discrete E. coli colonies. These
colonies were used to inoculate 1 mL liquid cultures to allow
expression of FAb fragments for use in screening experiments.
ELISA-Based Screening of FAbs for CD38 Binding
[0330] Each individual E. coli colony was used to express a FAb
that could be screened for CD38 ED-binding activity. Colonies were
inoculated into 1 mL starter cultures (supplemented with 100
.mu.g/mL carbenicillin and 2% glucose) in 96-well deep-well plates
(Costar) and incubated overnight at 37.degree. C. with shaking at
350 rpm (Innova R44 shaker; 1 inch orbit). These starter cultures
were diluted 1:100 into a 1 mL expression culture (2YT supplemented
with 100 .mu.g/mL carbenicillin) and grown to an optical density
(600 nm) of 0.5-0.8. FAb expression was induced by adding
isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final
concentration of 1 mM. Cultures were incubated at 25.degree. C. for
16 hrs.
[0331] FAb samples were prepared by harvesting cells by
centrifugation (2,000 g, 10 mins) and performing a lysozyme
extraction. The cell pellet was resuspended in 200 .mu.L of lysis
buffer (160 .mu.g/mL lysozyme, 10 .mu.g/mL RNase A, 5 .mu.g/mL
DNase and complete protease inhibitors (Roche, Nutley, N.J.)) and
shaken at 400 rpm for 30 minutes at 21.degree. C. Following
addition of a further 100 .mu.l of lysis buffer, the reactions were
incubated for a further 30 minutes as described previously.
Clarified lysates were isolated following centrifugation at 3,000 g
for 10 minutes and stored at 4.degree. C. until required.
[0332] To screen by enzyme-linked immunosorbent assay (ELISA) for
human CD38 ED-binders derived from the phage display biopanning,
human CD38 extracellular domain (ED) (produced in HEK 293-6E cells
and biotinylated as described above) was captured on
streptavidin-coated ELISA plates (Nunc) at 1 .mu.g/mL. Plates were
then washed and individual FAb samples (prepared as described
above) were added to individual wells on the ELISA plates. FAbs
were allowed to bind the captured CD38 ED for an hour at room
temperature and then washed three times with PBS-T (1.times.PBS
supplemented with 0.1% Tween.RTM.20). FAbs that bound to CD38 ED
were detected by incubation for 30 minutes at room temperature with
an anti-V5-HRP conjugated antibody (Invitrogen, Carlsbad, Calif.)
to detect the V5 tag fused to the C-terminus of the FAb heavy
chain. Plates were washed to remove unbound antibody and the assay
signal developed by incubation with 50 .mu.L
3,3',5,5'-Tetramethylbenzidine (Sigma-Adrich, St. Louis, Mo.) and
quenching with 50 .mu.L 1 M HCl. Assay signals were read at A450 nm
using a microplate reader (BMG Labtech). Results were expressed as
the raw A450 nm value, where any signal 2-fold greater than the
average assay background was defined as `positive`.
[0333] In later assays FAb cross-reactivity with cynomolgus monkey
CD38 ED was assessed by coating biotinylated cynomolgus monkey CD38
ED onto streptavidin coated ELISA plates and proceeding as
described above. Plasmids encoding FAbs cross-reactive with both
human and cynomolgus monkey CD38 ED were isolated and sequenced. Of
approximately 1,000 FAbs screened for binding to human and
cynomolgus monkey CD38 ED, six genetically unique FAbs were
identified. Table 21 summarises the FAb sequence data obtained. The
variable regions of some of these antibodies are shown in FIG.
13.
TABLE-US-00024 TABLE 21 Campaign Number FAb name V.sub.H sequence
V.sub.K/V.sub.L sequence 1 X910/12 SEQ ID NO: 395 SEQ ID NO: 394 1
X913/15 SEQ ID NO: 397 SEQ ID NO: 396 2 X355/01 SEQ ID NO: 421 SEQ
ID NO: 420 2 X355/02 SEQ ID NO: 391 SEQ ID NO: 390 2 X355/04 SEQ ID
NO: 423 SEQ ID NO: 422 2 X355/07 SEQ ID NO: 393 SEQ ID NO: 392
[0334] All FAbs were converted into IgG1 format by cloning into the
pTT5 vectors (described above), expressed in HEK293-6E cells and
the resulting IgGs purified by protein A affinity chromatography as
described above.
Assessing Binding of IgGs to Human CD38 Positive Cell Line
RPMI-8226
[0335] The ability of the phage derived antibodies to bind the
model human CD38 positive myeloma cell line RPMI-8226 (obtained
from the Health Protection Agency Culture Collections, Porton Down,
Salisbury, SP4 OJG, UK) in flow cytometry-based assays was tested.
Briefly, viable RPMI-8226 cells (2.times.10.sup.5, as judged by
trypan blue exclusion) were incubated with each antibody or with a
human IgG.sub.1 isotype control antibody preparation
(Sigma-Aldrich, St. Louis, Mo.) at various concentrations in 100
.mu.l of FACS buffer (PBS plus 1% fetal calf serum, FCS) in 96 well
plates for 20 minutes on ice in the dark. Cells were washed twice
with FACS buffer before incubation for 20 minutes in 100 .mu.l of
FACS buffer containing goat anti-human IgG (Fc-specific, conjugated
to fluorescein isothiocyanate, FITC; Sigma-Aldrich, St. Louis,
Mo.). After washing with FACS buffer, cells were resuspended in
FACS buffer and analysed for antibody binding by flow cytometry on
a FACS Canto (BD Biosciences, San Diego, Calif.) using EV, side
scatter and FL-1 gating. Results are expressed as mean fluorescent
intensity (MFI) plotted against protein concentration (FIG.
14).
Generation of Anti-CD38 Antibodies by Genetic Immunization
[0336] Monoclonal antibodies against human CD38 ED were generated
by genetic immunization with corresponding conventional protein
immunization of rats. For genetic immunization, the DNA sequence of
human CD38 ED is provided in SEQ ID NO:129. The corresponding
conceptually translated protein sequence is given in SEQ ID NO:130.
The DNA sequence of SEQ ID NO:129 was cloned into a plasmid for
genetic immunization using restriction enzyme technology.
Expression of the resulting plasmid allowed the secretion of
soluble CD38 ED tagged by a c-myc epitope at the N- or C-terminus.
The c-myc epitope was utilized to confirm expression of CD38
ED.
[0337] Rats were then immunized six times with the plasmid using a
Helios gene gun (Bio-Rad, Germany) according to a published
procedure (Kilpatrick et al., Hybridoma 17: 569-576, 1998). One
week after the last application of the immunization plasmid, each
rat was boosted by intradermal injection of untagged recombinant
human CD38 ED. Untagged human CD38 ED for this purpose was produced
by removing the protein tags from SEQ ID NO:127 by thrombin
cleavage followed by purification over a size exclusion column.
[0338] Four days later, the rats were sacrificed and their
lymphocytes fused with myeloma cells using polyethylene glycol
(HybriMax.TM.; Sigma-Aldrich, Germany), seeded at 100,000 cells per
well in 96-well microtiter plates and grown in DMEM medium
supplemented with 10% fetal bovine serum and HAT additive for
hybridoma selection (Kilpatrick et al., 1998, supra).
Screening Hybridoma Supernatants for Human and Cynomolgus Monkey
CD-38 Cross-Reactivity
[0339] Duplicate 100 .mu.L samples of each hybridoma supernatant
were coated onto separate wells of a maxisorp ELISA plate (Nunc
Plasticware, Thermo Scientific, Rochester, N.Y. 14625, USA) through
incubation at room temperature for an hour. Plates were washed
three times in 1.times.PBS-T and subsequently blocked by addition
of 2% BSA/1.times.PBS. Following incubation for 1 hour at room
temperature, plates were washed as described previously. To one
well of each rat antibody duplicate well was added 0.1 .mu.g of
biotinylated human CD38 in a final volume of 100 .mu.L 1.times.PBS.
To the second well of each rat antibody duplicate well was added
0.1 .mu.g of biotinylated cynomolgus monkey CD38 ED in a final
volume of 100 .mu.L 1.times.PBS. Plate wells were washed as
described previously prior to detection of bound biotinylated CD38
ED using a Streptavidin-HRP conjugate (BD Biosciences, San Diego,
Calif.). Plates were washed as above to remove unbound
Streptavidin-HRP conjugate and the assay signal developed by
incubation with 50 .mu.L 3,3',5,5'-Tetramethylbenzidine
(Sigma-Aldrich) and quenching with 50 .mu.L 1 M HCl. Assay signals
were read at A.sub.450 nm using a microplate reader (BMG Labtech,
Cary, N.C.). Of the 15 hybridoma supernatants tested, all fifteen
bound human CD38 ED and seven bound cynomolgus monkey CD38 ED
(Table 22) as determined by ELISA. The cross-reactive antibodies
are referred to as R5D1, R7F11, R5E8, R10A2, R10B10, R3A6 and
R7H11.
Flow Cytometry Binding of Rat Antibodies to Human CD38 Positive
Cell Line RPMI-8226
[0340] Viable RPMI-8226 cells (2.times.10.sup.5, as judged by
trypan blue exclusion) were incubated with 100 .mu.L of rat
hybridoma supernatant for 20 minutes on ice in the dark. Cells were
washed twice with FACS buffer (1.times.PBS plus 1% FCS) before
incubation for 20 minutes in 100 .mu.l of FACS buffer containing
anti-rat IgG-FITC conjugate (Sigma-Aldrich). After washing cells in
FACS buffer, they were resuspended in FACS buffer and analysed for
antibody-binding by flow cytometry on a FACS Canto (BD Biosciences,
San Diego, Calif.) using EV, side scatter and FL-1 gating. Results
were expressed as mean fluorescent intensity (MFI). Of the 15 rat
antibodies exhibiting positive binding to human CD38 ED by ELISA,
five showed weak or negligible binding to CD38 expressed on the
human myeloma cell line RPMI-8226 by FACS (Table 22).
TABLE-US-00025 TABLE 22 Binding to Cynomolgus FACS binding to Rat
Binding to Human monkey RPMI-8226 cells antibody CD38 ED (ELISA)
CD38 ED (ELISA) (MFI) R3A6 Y Y 279 R5D1 Y Y 12207 R5E8 Y Y 10618
R7F4 Y N 310 R7F11 Y Y 11897 R7H11 Y Y 680 R8A7 Y N 5994 R9B6 Y N
146 R9C7 Y N 143 R9C10 Y N 645 R9E5 Y N 179 R9G5 Y N 2717 R10A2 Y Y
4470 R10A9 Y N 12807 R10B10 Y Y 858
FACS Binding Background MFI Average was 153
Molecular Characterisation of Rat Antibodies
[0341] Six rat antibody hybridomas--R5D1, R7F11, R5E8, R10A2,
R10B10 and R7H11--were selected for molecular characterization. RNA
extraction from pelleted hybridoma cells of each clone was
performed using TRI reagent (Sigma-Aldrich, St. Louis, Mo.)
according to manufacturer's directions. The variable regions of
each antibody were amplified using Rapid Amplification of cDNA Ends
(RACE) reverse transcription polymerase chain reaction (RT-PCR)
methodology according to manufacturer's directions (Clontech
[Mountain View, Calif.] SMART RACE kit; Ambion Life Technologies
[Foster City, Calif.] RLM-RACE kit). Gene-specific reverse PCR
primers to amplify the rat heavy chain variable domains by 5'-RACE
were designed to anneal to the available rat heavy chain constant
region sequences. Similarly, gene specific reverse PCR primers to
amplify the rat light chains were designed to anneal to the rat
kappa chain constant region sequences, while further primers were
designed to anneal to the rat lambda chain constant region
sequences.
[0342] 5'-RACE PCR was performed according to manufacturer's
directions (Life Technologies; Clontech) using PfuUltraII
polymerase (Agilent). Following 5'-RACE PCR, products were
separated by agarose gel electrophoresis and bands of approximately
the predicted size based on the location of the reverse primer in
the constant region were excised from the gels. DNA was purified
from agarose gel slices using a Qiaquick spin gel extraction kit
(Qiagen) according to manufacturer's instructions. Insert DNA was
cloned and propagated in E. coli using a StrataClone Blunt PCR
Cloning Kit (Agilent, Santa Clara, Calif.) according to
manufacturer's instructions. Single colonies from transformations
were cultured and plasmid DNA prepared using a GenElute.TM. plasmid
miniprep kit (Sigma-Aldrich, St. Louis, Mo.). DNA inserts were
sequenced and antibody variable regions identified in the
conceptually translated protein sequences.
[0343] Vectors were constructed using the rat antibody variable
region sequences grafted onto human IgG1 constant sequences for the
heavy chain variable region and, human kappa or lambda backbones
(keeping the same light chain isotype as in the rat antibodies).
The resulting variable region sequences of each clone are listed in
Table 23. Subsequent co-expression of the corresponding heavy- and
light chains in HEK293-6E cells, in the context of the pTT5
vectors, was followed by protein A purification of the resulting
IgGs as described above.
TABLE-US-00026 TABLE 23 Light chain Rat antibody isotype Heavy
chain (VH) Light chain (VL) R5D1 Kappa SEQ ID NO: 399 SEQ ID NO:
398 R5E8 Kappa SEQ ID NO: 401 SEQ ID NO: 400 R10A2 Kappa SEQ ID NO:
403 SEQ ID NO: 402 R10B10 Lambda SEQ ID NO: 425 SEQ ID NO: 424
R7H11 Lambda SEQ ID NO: 427 SEQ ID NO: 426 R7F11 kappa SEQ ID NO:
429 SEQ ID NO: 428
Affinity of Anti-CD38 Antibodies for Human and Cynomolgus Monkey
CD38
[0344] The binding affinities of a selection of the antibodies
produced against human and cynomolgus monkey CD38 were measured.
Briefly, using a Biacore T200, Protein A was immobilized onto Flow
Cell (FC) 1 (FC1) and FC2 (or alternatively FC3 and FC4) of a CM5
research grade sensor chip using amine coupling, giving
approximately 2000 RU. FC1 was used as a blank throughout the
experiments. The experiments were run in HBS-P buffer (0.01 M HEPES
pH 7.4, 0.15 M NaCl, 0.005% v/v Surfactant P20). At a flow rate of
20 .mu.l/min, 20 .mu.l of 5 .mu.g/mL of antibody was passed over
FC2. Human CD38 ED or separately, cynomolgus monkey CD38 ED was
passed over the surface of FC1 and FC2 at concentrations ranging
from 25 nM to 200 nM. Regeneration of the surface was performed
using 10 mM Glycine, pH 1.0. The FC1 sensorgram data was subtracted
from FCS and the curves were fitted using a 1:1 Langmuir equation
to generate the k.sub.d, k.sub.a and K.sub.D values. This data
shows that cross-reactivity for human and cynomolgus monkey CD38 is
maintained on conversion of the human phage-derived Fabs into human
IgGs and rat antibodies into chimeric rat-human IgGs (Table
24).
TABLE-US-00027 TABLE 24 CD38 Antibody Ligand ka (1/Ms) .times.
10.sup.5 kd (1/s) K.sub.D (nM) X355/02 IgG1 Human 1.18 0.000892 7.6
X355/02 IgG1 Cynomolgus 0.834 0.002282 27.4 X355/07 IgG1 Human 1.15
0.00132 11.4 X355/07 IgG1 Cynomolgus 11.3 0.01375 12.2 R10A2 IgG1
Human 6.6 0.0004.79 0.7 R10A2 IgG1 Cynomolgus 8.98 0.001928 2.2
R5D1 IgG1 Human 2.43 0.000239 1.0 R5D1 IgG1 Cynomolgus 11.1
0.001102 1.0 R5E8 IgG1 Human 4.05 0.00118 2.9 R5E8 IgG1 Cynomolgus
4.52 0.001898 4.2
Anti-CD38-Attenuated IFN Fusion Protein Constructs
[0345] To determine whether the surprising result obtained with an
anti-CD20 antibody fused to an attenuated IFN.alpha. could be
replicated with other antibodies, and in particular antibodies
targeting an antigen unrelated to CD20, fusion protein constructs
comprising the fully human IgG1:kappa anti-CD38 antibody G005
(composed of SEQ ID NOS:135 (heavy chain) and 134 (light chain))
and IFN.alpha. (SEQ ID NO:3), with or without various attenuating
mutations was made. FIG. 15 shows the results of the "off target
assay" (as described above) using the iLite kit. Because faint CD38
signal was observed on the iLite cell line by flow cytometry (not
shown), the CD38 antigen was blocked by the addition of excess
naked (e.g. without IFN or IFN variants fused to it) anti-CD38
antibody for all iLite experiments using anti-CD38-IFN fusion
protein constructs; in each case, the concentration of blocking
naked CD38 antibody used was 0.5 mg/ml. Also in each case, the same
antibody clone being assayed as an IFN or IFN-variant fusion
protein construct was used to block any interaction with CD38.
[0346] FIG. 15 shows the off target activity of free wild type
IFN.alpha.2b (IFN.alpha.) (SEQ ID NO:3) vs. wild type IFN.alpha.2b
fused to the C-terminus of the CD38 antibody G005 (De Weers et al.
(U.S. Pat. No. 7,829,673). The latter fusion protein construct
(G005-HC-L0-IFN.alpha. IgG4) was of IgG4:kappa isotype and had no
intervening linker between the C-terminus of the heavy chain and
the first residue of the IFN.alpha. and is described by SEQ ID
NOS:150 (heavy chain) and 134 (light chain). As illustrated in FIG.
15, the anti CD38 antibody-non-attenuated IFN.alpha.2b fusion
protein construct was 27-fold less potent (19.5/0.726=27) than free
IFN.alpha.2.beta. in the off-target assay (e.g. in the absence of
CD38-targeting). FIG. 16 shows a comparison between the same two
constructs in the "on target (ARP1) assay", in which the anti-CD38
antibody was allowed to bind to CD38, which was expressed at high
levels on the ARP-1 cell line. The G005-HC-L0-IFN.alpha. IgG4
fusion protein construct was 3.6-fold (14.7/4.08=3.6) more potent
than free IFN.alpha.2b, presumably due to the targeted delivery of
the IFN to the CD38.sup.+ myeloma cells. Therefore, the
G005-HC-L0-IFN.alpha. IgG4 fusion protein construct has an antigen
specificity index (ASI) of 97 (27.times.3.6=97; Table 25).
TABLE-US-00028 TABLE 25 Antigen Specificity EC50 IFN.alpha./ Index
EC50 On EC50 TA EC50 Off EC50 IFN.alpha./ Column Test Article
Target (On Target; Target (pM) EC50 TA (Off 3/ (TA) (pM) ARP-1
ARP-1) iLite Target; iLite) Column 5 IFN.alpha. 10.8 1.00 0.260
1.00 1.00 G005-HC-L0- 3.6* 0.037** 97 IFN.alpha. IgG4 G005-HC-L0-
186 0.0581 25,800 1.01 .times. 10.sup.-5 5,750 IFN.alpha. (R144A)
IgG4 G005-HC-L0- 290 0.0372 1.08 .times. 10.sup.5 2.41 .times.
10.sup.-6 15,400 IFN.alpha. (R144S) IgG4 G005-HC-L0- ND ND
>10.sup.5 ND ND IFN.alpha. (R144E) IgG4 G005-HC-L0- ND ND 9,970
2.61 .times. 10.sup.-5 ND IFN.alpha. (R144G) IgG4 G005-HC-L0- ND ND
1,690 1.54 .times. 10.sup.-4 ND IFN.alpha. (R144H) IgG4 G005-HC-L0-
ND ND <100 ND ND IFN.alpha. (R144K) IgG4 G005-HC-L0- ND ND 431
0.000603 ND IFN.alpha. (R144N) IgG4 G005-HC-L0- ND ND 3,500 7.43
.times. 10.sup.-5 ND IFN.alpha. (R144Q) IgG4 G005-HC-L0- 333 0.0324
30,800 8.44 .times. 10.sup.-6 3,840 IFN.alpha. (R144T) IgG4
G005-HC-L0- 306 0.0353 92,100 2.82 .times. 10.sup.-6 12,500
IFN.alpha. (R144Y) IgG4 G005-HC-L0- 257 0.0420 1.59 .times.
10.sup.5 1.64 .times. 10.sup.-6 25,600 IFN.alpha. (R144I) IgG4
G005-HC-L0- 191 0.0565 26,700 9.74 .times. 10.sup.-6 5,800
IFN.alpha. (R144L) IgG4 G005-HC-L0- ND ND 86,900 2.99 .times.
10.sup.-6 ND IFN.alpha. (R144V) IgG4 G005-HC-L0- 23.8 0.454 2,040
0.000127 3,570 IFN.alpha. (A145G) IgG4 G005-HC-L0- 222 0.0486
52,600 4.94 .times. 10.sup.-6 9,840 IFN.alpha. (A145D) IgG4
G005-HC-L0- ND ND <100 ND ND IFN.alpha. (A145E) IgG4 G005-HC-L0-
113 0.0956 24,900 1.04 .times. 10.sup.-5 9,190 IFN.alpha. (A145H)
IgG4 G005-HC-L0- ND ND 28.9 0.00900 ND IFN.alpha. (A145I) IgG4
G005-HC-L0- 174 0.0621 6.62 .times. 10.sup.5 3.93 .times. 10.sup.-7
1.58 .times. 10.sup.5 IFN.alpha. (A145K) IgG4 G005-HC-L0- ND ND 239
0.00109 ND IFN.alpha. (A145L) IgG4 G005-HC-L0- ND ND 309 0.000841
ND IFN.alpha. (A145N) IgG4 G005-HC-L0- ND ND 709 0.000367 ND
IFN.alpha. (A145Q) IgG4 G005-HC-L0- ND ND >10.sup.6 ND ND
IFN.alpha. (A145R) IgG4 G005-HC-L0- ND ND <2 ND ND IFN.alpha.
(A145T) IgG4 G005-HC-L0- 91.4 0.118 19,200 1.35 .times. 10.sup.-5
8,740 IFN.alpha. (A145Y) IgG4
[0347] In order to determine whether the ASI could be increased, as
was observed for the anti-CD20-IFN.alpha. fusion protein
constructs, several variants were constructed by attenuating the
IFN portion of the anti-CD38-IFN.alpha. fusion protein construct by
mutation. Numerous different attenuating mutations were made in the
context of the G005 or other CD38 monoclonal antibodies. In
addition, constructs of different IgG isotypes (IgG1 and IgG4) and
linker lengths (L0, no linker; L6, 6 amino acid linker (SGGGGS, SEQ
ID NO:132)) were made. The off-target assay and two types of
on-target assays (using Daudi and ARP-1), both described in detail
above, were run and the results are shown in FIGS. 17-38 and
tabulated in Tables 25-33. The discussion below summarizes these
results with references to the data in these tables (all of which
is derived from FIGS. 17-38).
[0348] Table 26 characterizes the CD38 antibody G005, fused in
various configurations via the C-terminus of the heavy chain to
IFN.alpha. with the R144A attenuating mutation. Examples in this
table are of IgG1 and IgG4 isotype and either have no linker
between the antibody heavy chain and the IFN, L0, or have an
intervening 6 amino acid linker, L6 (composed of SEQ ID NOS:138,
140, 152, 146 (heavy chain) each combined with 134 (light chain)).
In all cases, the fusion protein constructs had dramatically
reduced potency on antigen-negative iLite cells (a reduction of
from 8,300 to 100,000 fold compared to free IFN.alpha.), but
substantially maintained the potency exhibited by free IFN.alpha.
on CD38 positive cells (Daudi). The G005-HC-L0-IFN.alpha. (R144A)
IgG4 construct (composed of SEQ ID NOS:152 (heavy chain) and 134
(light chain)), for example, has a 10.sup.5-fold lower potency than
free, wild type IFN.alpha. on antigen negative cells but its
potency is reduced only 3.5-fold (2.7/0.77=3.5) vs. free, wild type
IFN.alpha. on antigen positive cells (Table 26). This gives an
Antigen Specificity Index (ASI) of 29,000 for this fusion protein
construct.
TABLE-US-00029 TABLE 26 Antigen EC50 On EC50 IFN.alpha./ EC50
IFN.alpha./ Specificity Target EC50 TA EC50 Off EC50 TA Index (pM)
(On Target; Target (pM) (Off Target; Column 3/ Test Article (TA)
Daudi Daudi) iLite iLite) Column 5 IFN.alpha. 0.77 1.0 0.30 1.0 1.0
G005-HC-L6- 1.3 0.59 11,000 2.7 .times. 10.sup.-5 22,000 IFN.alpha.
(R144A) IgG4 G005-HC-L0- 2.7 0.29 30,000 1.0 .times. 10.sup.-5
29,000 IFN.alpha. (R144A) IgG4 G005-HC-L6- 1.9 0.41 2,600 0.00012
3,400 IFN.alpha. (R144A) IgG1 G005-HC-L0- 7.3 0.11 6,800 4.4
.times. 10.sup.-5 2,500 IFN.alpha. (R144A) IgG1
[0349] Table 27 shows examples using another IFN.alpha. attenuating
mutation, A145G, as a construct with the same G005 antibody in
either IgG1 or IgG4 isotypes, with either no linker or the L6
linker (composed of SEQ ID NOS: 142, 144, 148 (heavy chain) each
combined with SEQ ID 134 (light chain)). The G005-HC-L6-IFN.alpha.
(A145G) IgG4 construct (composed of SEQ ID NOS:148 (heavy chain)
and 134 (light chain)), for example, showed an ASI of 20,000.
TABLE-US-00030 TABLE 27 Antigen Specificity EC50 On EC50
IFN.alpha./ EC50 IFN.alpha./ Index Target EC50 TA EC50 Off EC50 TA
Column (pM) (On Target; Target (pM) (Off Target; 3/ Test Article
(TA) Daudi Daudi) iLite iLite) Column 5 IFN.alpha. 0.48 1.0 0.087
1.0 1.0 G005-HC-L0- 0.74 0.65 510 0.00017 3,800 IFN.alpha. (A145G)
IgG1 G005-HC-L6- 1.0 0.48 730 0.00012 4,000 IFN.alpha. (A145G) IgG1
G005-HC-L6- 0.59 0.81 2200 4.0 .times. 10.sup.-5 20,000 IFN.alpha.
(A145G) IgG4
[0350] Table 28 shows examples in which the mutated IFN.alpha. is
attached to the light chain rather than the heavy chain, with
either no intervening linker or the L6 linker (composed of SEQ ID
NOS:210 or 208 (light chain), respectively, each combined with SEQ
ID NO:135 (heavy chain)). In both cases, the fusion protein
constructs demonstrated a high ASI of 5,900 and 7,200,
respectively.
TABLE-US-00031 TABLE 28 Antigen Specificity EC50 IFN.alpha./ EC50
IFN.alpha./ Index EC50 On EC50 TA EC50 Off EC50 TA Column Test
Article Target (pM) (On Target; Target (pM) (Off Target; 3/ (TA)
Daudi Daudi) iLite iLite) Column 5 IFN.alpha. 1.1 1.0 0.21 1.0 1.0
G005-LC-L6- 8.5 0.13 12,000 1.8 .times. 10.sup.-5 7,200 IFN.alpha.
(A145G) IgG1 G005-LC-L0- 21 0.052 24,000 8.8 .times. 10.sup.-6
5,900 IFN.alpha. (A145G) IgG1
[0351] Tables 29 and 30 demonstrate the ASI for the same fusion
protein constructs but use an alternative cell line (ARP-1, a
myeloma) for determining activity on CD38.sup.+ cells. Using this
method, the ASIs for these fusion protein constructs ranged from
1,200-55,000.
TABLE-US-00032 TABLE 29 Antigen EC50 IFN.alpha./ Specificity EC50
On EC50 TA EC50 Off EC50 IFN.alpha./ Index Test Article Target (On
Target; Target (pM) EC50 TA (Off Column 3/ (TA) (pM) ARP-1 ARP-1)
iLite Target; iLite) Column 5 IFN.alpha. 6.0 1.0 0.30 1.0 1.0
G005-HC-L6- 28 0.21 11,000 2.7 .times. 10.sup.-5 7,800 IFN.alpha.
(R144A) IgG4 G005-HC-L0- 85 0.071 30,000 1.0 .times. 10.sup.-5
7,100 IFN.alpha. (R144A) IgG4 G005-HC-L6- 21 0.29 2,600 0.00012
2,400 IFN.alpha. (R144A) IgG1 G005-HC-L0- 110 0.054 6,800 4.4
.times. 10.sup.-5 1,200 IFN.alpha. (R144A) IgG1
TABLE-US-00033 TABLE 30 Antigen EC50 IFN.alpha./ Specificity EC50
On EC50 TA EC50 Off EC50 IFN.alpha./ Index Test Article Target (On
Target; Target EC50 TA (Off Column 3/ (TA) (pM) ARP-1 ARP-1) (pM)
iLite Target; iLite) Column 5 IFN.alpha. 9.5 1.0 0.087 1.0 1.0
G005-HC-L0- 8.0 1.2 510 0.00017 7,100 IFN.alpha. (A145G) IgG1
G005-HC-L6- 4.0 2.4 730 0.00012 20,000 IFN.alpha. (A145G) IgG1
G005-HC-L6- 4.4 2.2 2200 4.0 .times. 10.sup.-5 55,000 IFN.alpha.
(A145G) IgG4
[0352] Numerous other examples of mutated versions of IFN.alpha.,
in the context of the G005-HC-L0-IFN.alpha. IgG4 fusion protein
construct are set out in Table 25. The majority of these mutants
(R144 mutated to A, S, E, G, H, N, Q, T, Y, I, L or V (composed of
SEQ ID NOS:152, 172, 156, 158, 160, 168, 170, 174, 178, 162, 166,
176 (heavy chain), respectively, each combined with SEQ ID NO:134
(light chain)) and A145 mutated to G, D, H, I, K, L, N, Q, R or Y
(SEQ ID NOS:184, 180, 186, 188, 190, 192, 194, 196, 198, 206 (heavy
chain), respectively, each combined with SEQ ID NO:134 (light
chain)) showed significant attenuation of IFN.alpha. activity
compared to free wild type IFN.alpha. In this context, the A145V
and A145S mutants did not show appreciable attenuation. Any of
these point mutated, attenuated versions of IFN.alpha. could be
used in the context of the present invention as antibody fusion
protein constructs. Certain IFN variants may be preferred due to
showing higher ASIs. Other considerations, such as expression
level, immunogenicity, biophysical characteristics, etc., may also
be considered in evaluating constructs for optimal utility. Of the
numerous IFN.alpha. variants described in this document, those
shown here to yield a high ASI in the context of an antibody-fusion
protein construct include R144A, R144S, R144T, R144Y, R144I, R144L,
R145G, R145D, R145H, R145Y (Table 25), R33A+YNS (as illustrated in
the construct comprising SEQ ID NOS:286 (heavy chain) and 276
(light chain)), R33A (as illustrated in the construct comprising
SEQ ID NOS:436 (heavy chain) and 276 (light chain)) and R144A+YNS
(such as as illustrated in the construct comprising SEQ ID NOS:288
(heavy chain) and 276 (light chain)).
[0353] The mutation of A145D in the construct of SEQ ID NOS:180
(heavy chain) and 134 (light chain) compared to the A145E mutation
in the construct of SEQ ID NO:182 (heavy chain) and 134 (light
chain) produced unexpected results. Although both constructs have
similar amino acid sequences, differing by only a single methylene
group, they showed dramatically different effects on the
non-targeted activity of the IFN.alpha.. The A145E mutation had
minimal impact on the IFN.alpha. activity, however, the A145D
mutation drastically reduced activity (by 20,000-fold) and resulted
in a construct with high ASI (9,840).
[0354] Other examples of anti-CD38 antibody-attenuated IFN.alpha.
fusion protein constructs are shown in Tables 31-33. In addition to
the G005 antibody constructs, these tables show the on-target
activity, the off-target activity, and the ASI for anti-CD38
antibody-attenuated IFN.alpha. fusion protein constructs based on
certain novel antibodies. Fusion protein constructs of these
antibodies with the IFN.alpha. A145D or R144A mutations include the
following:
[0355] X910/12-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:248 (heavy chain) and SEQ ID NO:242 (light chain);
[0356] X910/12-HC-L0 IFN.alpha. (R144A) IgG4 composed of SEQ ID
NOS:246 (heavy chain) and SEQ ID NO:242 (light chain);
[0357] X913/15-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:256 (heavy chain) and SEQ ID NO:250 (light chain);
[0358] X913/15-HC-L0 IFN.alpha. (R144A) IgG4 composed of SEQ ID
NOS: 254 (heavy chain) and SEQ ID NO:250 (light chain);
[0359] X355/02-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:232 (heavy chain) and SEQ ID NO:226 (light chain);
[0360] X355/02-HC-L0 IFN.alpha. (R144A) IgG4 composed of SEQ ID
NOS:230 and SEQ ID NO:226 (light chain);
[0361] X355/07-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:240 (heavy chain) and SEQ ID NO:234 (light chain);
[0362] X355/07-HC-L0 IFN.alpha. (R144A) IgG4 composed of SEQ ID
NOS:238 and SEQ ID NO:234 (light chain);
[0363] R5D1-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:262 (heavy chain) and 258 (light chain);
[0364] R5E8-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:268 (heavy chain) and 264 (light chain); and
[0365] R10A2-HC-L0 IFN.alpha. (A145D) IgG4 composed of SEQ ID
NOS:274 (heavy chain) and 270 (light chain).
[0366] These fusion protein constructs all showed high ASIs,
ranging from 3,820 (X910/12-HC-L0-IFN.alpha. (R144A) IgG4) to
166,000 (X355/02-HC-L0-IFN.alpha. (A145D) IgG4).
TABLE-US-00034 TABLE 31 Antigen EC50 On EC50 IFN.alpha./
Specificity Target EC50 TA EC50 Off EC50 IFN.alpha./ Index Test
Article (pM) (On Target; Target EC50 TA (Off Column 3/ (TA) ARP-1
ARP-1) (pM) iLite Target; iLite) Column 5 IFN.alpha. 8.91 1.00
0.499 1.00 1.00 G005-HC-L0- 220 0.0405 73,200 6.82 .times.
10.sup.-6 5,940 IFN.alpha. (R144A) IgG4 X910/12-HC- 191 0.0466
41,000 1.22 .times. 10.sup.-5 3,820 L0-IFN.alpha. (R144A) IgG4
X913/15-HC- 84.1 0.106 29,600 1.69 .times. 10.sup.-5 6,270
L0-IFN.alpha. (R144A) IgG4 X355/02-HC- 147 0.0606 70,500 7.08
.times. 10.sup.-6 8,560 L0-IFN.alpha. (R144A) IgG4 X355/07-HC- 61.2
0.146 70,300 7.10 .times. 10.sup.-6 20,600 L0-IFN.alpha. (R144A)
IgG4
TABLE-US-00035 TABLE 32 Antigen EC50 On EC50 IFN.alpha./ EC50
IFN.alpha./ Specificity Target EC50 TA EC50 Off EC50 TA Index Test
Article (pM) (On Target; Target (pM) (Off Target; Column 3/ (TA)
ARP-1 ARP-1) iLite iLite) Column 5 IFN.alpha. 43.0 1.00 0.304 1.00
1.00 G005-HC-L0- 85.7 0.502 47,100 6.44 .times. 10.sup.-6 78,000
IFN.alpha. (A145D) IgG4 X910/12-HC- 336 0.128 22,500 1.35 .times.
10.sup.-5 9480 L0-IFN.alpha. (A145D) IgG4 X913/15-HC- 68.1 0.631
31,800 9.59 .times. 10.sup.-6 65,800 L0-IFN.alpha. (A145D) IgG4
X355/02-HC- 46.0 0.935 53,900 5.64 .times. 10.sup.-6 1.66 .times.
10.sup.-5 L0-IFN.alpha. (A145D) IgG4 X355/07-HC- 61.9 0.695 61,800
4.92 .times. 10.sup.-6 1.41 .times. 10.sup.-5 L0-IFN.alpha. (A145D)
IgG4
TABLE-US-00036 TABLE 33 Antigen EC50 On EC50 IFN.alpha./
Specificity Target EC50 IFN.alpha./ EC50 Off EC50 TA Index Test
Article (pM) EC50 TA (On Target (Off Target; Column 3/ (TA) ARP-1
Target; ARP-1) (pM) iLite iLite) Column 5 IFN.alpha. 16.1 1.00
0.271 1.00 1.00 R5D1-HC-L0- 51.3 0.314 49,500 5.48 .times.
10.sup.-6 57,300 IFN.alpha. (A145D) IgG4 R5E8-HC-L0- 89.2 0.180
38,400 7.06 .times. 10.sup.-6 25,500 IFN.alpha. (A145D) IgG4
R10A2-HC-L0- 32.7 0.492 29,600 9.16 .times. 10.sup.-6 53,700
IFN.alpha. (A145D) IgG4 X355/02-HC- 104 0.155 81,600 3.32 .times.
10.sup.-6 46,700 L0-IFN.alpha. (A145D) IgG4
[0367] The examples above demonstrate that mutated, attenuated
forms of IFN.alpha., attached to antibodies targeting CD20 (SEQ ID
NO:430) or CD38 (SEQ ID NO:131), show orders of magnitude greater
potency in IFN signaling on antigen-positive target cells than on
antigen-negative off-target cells. The results below provide
further examples using antibodies that target the attenuated
IFN.alpha. to two other antigens: CD138 and class I MHC.
[0368] CD138 (SEQ ID NO:432), also called Syndecan-1, is a heparin
sulfate proteoglycan that is thought to function as an adhesion
molecule. It is expressed on most multiple myeloma cells
(Dhodapkar, Blood; 91: 2679, 1998). Fusion protein constructs
consisting of mutated, attenuated IFN.alpha. and the
CD138-targeting antibody nBT062 (Ikeda, Clin Can Res., 15:4028,
2009; USPTO #20090175863, composed of SEQ ID NOS:330 (heavy chain)
and 326 (light chain)) were generated. As shown in FIG. 39, this
fusion protein construct, like the anti-CD38-attenuated IFN.alpha.
fusion protein construct, showed much greater anti-proliferative
potency on multiple myeloma cells (ARP-1, on-target assay) than a
non-targeted, isotype fusion protein (based on the antibody 2D12).
FIG. 39 shows that a 28 pM concentration (4.sup.th highest
concentration tested) of nBT062-HC-L0-IFN.alpha. (A145D) shows
greater anti-proliferative activity on the ARP-1 myeloma cell line
than does 6 nM (highest concentration tested) of the
isotype-HC-L0-IFN.alpha. (A145D) protein.
[0369] Another antigen that has been described as a potential
target for antibody therapy to treat cancer is the Class I MHC (see
for example Stein, Leuk. Lymphhoma 52(2):273-84, 2011). In order to
determine whether it was possible to apply the present invention in
relation to this target, antibody W6/32 (Barnstable et al. (1978),
Cell 14:9-20), was obtained by from ATCC (HB95). This antibody
reacts with monomorphic determinants on human HLA A,B,C molecules.
The antibody variable regions were cloned and sequenced using SMART
RACE cDNA Amplification kit (Clontech, Mountain View, Calif.) and
Mouse Ig-Primer Sets (Novagen/EMD Chemicals, San Diego, Calif.).
The amino acid sequences of the heavy chain and light chain
variable regions are shown as SEQ ID NOS:411 and 410, respectively.
The chimeric version of HB95, with the murine variable regions and
human IgG4 kappa constant regions, fused to IFN.alpha. with the
A145D mutation (HB95-HC-L0-IFN.alpha. (A145D) IgG4, composed of SEQ
ID NOS:316 (heavy chain) and 312 (light chain)) was expressed, and
its activity was compared to an isotype control antibody fused in
the same way to the same IFN.alpha. mutant
(Isotype-HC-L0-IFN.alpha. (A145D) IgG4, where the isotype variable
regions were derived from antibody 2D12). The "on-target (ARP-1)"
assay was run as described above for the CD38-targeted antibodies
(ARP-1 is class I MHC-positive). The results are shown in FIG. 40a.
The class I MHC-targeted attenuated IFN.alpha. is orders of
magnitude more potent than the isotype control-attenuated
IFN.alpha. fusion protein construct on the same cells, coming
within about 9-fold (139/16=8.7) of the wild type IFN.alpha.. While
HB95-HC-L0-IFN.alpha. (A145D) IgG4 shows significant activity below
100 pM, the isotype-HC-L0-IFN.alpha. (A145D) IgG4 shows no
significant activity even at 6 nM.
[0370] FIG. 40b demonstrates that antibody fragments may substitute
for full-length antibodies and provide similar properties, namely
high ASIs. This figure shows the effects of various Fab-attenuated
IFN.alpha. fusion protein constructs on the proliferation of ARP-1
cells. Two non-ARP-1 targeted constructs,
"Palivizumab-HC-L6-IFN.alpha. (A145D) Fab" (composed of SEQ ID
NOS:298 (heavy chain) and 290 (light chain)) and
"2D12-HC-L6-IFN.alpha. (A145D) Fab" (composed of SEQ ID NOS:356
(heavy chain) and 344 (light chain)), show very low potency on this
cell line (EC50's from 2,410-17,000). By contrast, when the Fab
portion of the fusion protein construct does target a cell surface
antigen, in this case class I MHC, as for the fusion protein
construct "HB95-HC-L6-IFN.alpha. (A145D) Fab" (composed of SEQ ID
NOS:320 (heavy chain) and 312 (light chain)), the potency is even
higher than free, wild type IFN.alpha.. The antigen-targeted
attenuated construct is 2,760-19,450-fold more potent than the
non-targeted attenuated constructs.
Antiviral Activity of Targeted, Attenuated IFN.alpha.
[0371] The anti-viral activity of IFN.alpha. is well-known and
recombinant IFN.alpha. is an FDA-approved treatment for hepatitis C
viral infections. The effect of a host cell surface-targeted vs.
non-targeted antibody-attenuated IFN.alpha. fusion protein
construct on the cytopathic activity of the EMC virus on A549
cells, which are class I MHC-positive, was compared.
Methods:
[0372] IFN activity was measured using the cytopathic effect
inhibition (CPE) assay as described Rubinstein (J. Virol. 37,
755-8, 1981). Briefly, 10.sup.4 human adenocarcinoma A549 cells
(ATCC, Manassas, Kans.) per well were incubated with test sample or
IFN (human IFN-.alpha.2A) overnight. Cells were then challenged
with EMC virus for 48-56 hours, followed by staining with crystal
violet. A visual CPE determination was performed, followed by
solubilization of the crystal violet and absorbance measurement at
570 nm. Nonlinear regression analysis was performed using a
4-parameter sigmoidal fit with variable slope (GraphPad Prism). One
unit of IFN.alpha. activity is defined as the amount of interferon
required to reduce the cytopathic effect by 50%. The units are
determined with respect to the international reference standard for
human IFN.alpha.2, provided by the National Institutes of Health
(see Pestka, S. "Interferon Standards and General Abbreviations,"
in Methods in Enzymology (S. Pestka, ed.), Academic Press, New York
vol 119, pp. 14-23, 1986). The samples tested in this assay were
IFN.alpha. (Intron A, inverted triangles), Anti-MHC class I
targeted attenuated IFN.alpha. designated HB95-HC-L0-IFN.alpha.
(R145D) IgG4 (closed squares), and istoype control
(2D12)-attenuated IFN.alpha. (Isotype-HC-L0-IFN.alpha. (R145D)
IgG4; triangles). Data is plotted as viability vs IFN.alpha. molar
equivalents.
Results:
[0373] Results are shown in FIG. 41. In this assay, IFN.alpha.
protects A549 cells from virally induced cytopathic cell death
(CPE) as expected, showing at EC50 of 0.18 pM. Introducing the
R145D mutation to the IFN.alpha. (and attaching it to an antibody
that does not bind to the A549 cells) reduces its anti-viral
potency by 108,000-fold (19,461/0.18=108,167). By contrast, by
attaching the same mutant IFN to an A549-targeting antibody (HB95),
the potency is increase by 17,000-fold (19,461/1.15=16,923). This
corresponds to an ASI of 16,900 (19,461/1.15=16,922).
Targeted, Attenuated IFN.beta.
[0374] IFN.beta. also has been shown in numerous publications (see
above) to have anti-proliferative activity on various types of
cancer cells. A fusion protein construct between an anti-CD38
antibody (G005) and IFN.beta. (SEQ ID NO:91), G005-HC-L0-IFN.beta.
IgG4 (composed of SEQ ID NOS:212 (heavy chain) and 134 (light
chain)) as well as an identical construct but carrying a single
point mutation (R35A), known to reduce IFN.beta. potency (Runkel et
al. J. Biol. Chem. 273:8003-8 (1998), composed of SEQ ID NOS:214
(heavy chain) and 134 (light chain)) was therefore made. In both
constructs, the unpaired cysteine at position 17 of IFN.beta. was
mutated to a serine in order to improve expression yields and
product homogeneity. FIG. 42 shows the activity of these three
proteins under conditions where there is no antibody-assisted
targeting ("off target assay" using iLite kit). In this assay, the
attachment of an IgG onto the N-terminus of IFN.beta. attenuates
its potency by 72-fold (57.6/0.799=72). By making the R35A mutation
in this fusion protein construct, its potency is further reduced by
280-fold (16,100/57.6=280) so that it is 20,150-fold
(16,100/0.799=20,150) less potent than free, wild type IFN.beta..
In stark contrast, FIG. 43 shows the potency of these three
proteins under conditions in which the CD38 antibody can target the
IFN.beta. to cells is fairly similar. In this assay, the
antibody-attenuated IFN.beta. fusion protein construct
(G005-HC-L0-IFN.beta. (R35A) IgG4) is only 1.4-fold (46.9/32.7=1.4)
less potent than the antibody-non-attenuated IFN.beta. fusion
protein construct and only 4.5-fold (46.9/10.5=4.5) less potent
than free, wild type IFN.beta.. This data is summarized in Table
34. This demonstrates that the surprising finding that attenuating
mutations in an interferon that is part of an antibody-IFN fusion
protein construct can disproportionally affect non-targeted vs.
targeted cells, as observed for IFN.alpha. (Table 20), also holds
true for IFN.beta.. In the present example of the
anti-CD38-IFN.beta. fusion protein constructs, the attenuating
mutation reduced the potency by only 1.4-fold under conditions when
the antibody could direct the IFN to the target cells, vs. 280-fold
for cells in which the fusion protein construct could not target
the cell surface antigen. As a result, the antibody-attenuated
IFN.beta. fusion protein construct in the present example shows an
ASI of 4,630 (Table 34). The R147A mutation in IFN.beta., as an
alternative to the R35A mutation, was also found to produce
antibody-IFN.beta. fusion protein constructs with a significantly
greater ASI than free IFN.beta. (data not shown). The examples
below will show that this "selective attenuation" can also be
observed with ligands that are structurally unrelated to IFN.alpha.
and .beta., namely to IL-4 and IL-6.
TABLE-US-00037 TABLE 34 Antigen EC50 On EC50 IFN.beta./ EC50
IFN.beta./ Specificity Target EC50 TA EC50 Off EC50 TA Index Test
Article (pM) (On Target; Target (pM) (Off Target; Column 3/ (TA)
ARP-1 ARP-1) iLite iLite) Column 5 IFN.beta. 10.5 1.00 0.799 1.00
1.00 G005-HC-L0- 46.9 0.224 16,100 4.84 .times. 10.sup.-5 4,630
IFN.beta. (R35A) IgG4 G005-HC-L0- 32.7 0.321 57.6 0.0135 23.8
IFN.beta. IgG4
Interleukin-4 (IL-4)
[0375] IL-4 is a helical bundle cytokine with multiple
physiological activities, including the ability to bias T helper
cell development towards Th2 and away from Th1. Since Th1 cells
play a pathological role in certain autoimmune settings, it could
be therapeutically advantageous to use IL-4 to influence T helper
cell development away from Th1, i.e. to create a "Th1 diversion."
To avoid side effects related to IL-4's activity on other cell
types, it would be advantageous to attenuate IL-4's activity by
mutating it, and then attach it to an antibody that would direct it
to activated (preferentially recently activated) helper T cells.
The antibody chosen for this purpose was J110, a mouse anti-human
PD-1 clone described by Iwai et. al. (Immunol Lett. 83:215-20,
2002). PD-1 (SEQ ID NO:431) is expressed on recently activated Th0
cells.
[0376] The J110 antibody (murine variable regions and human
IgG1:kappa constant regions; the amino acid sequences of J110 heavy
and light chain variable regions are shown as SEQ ID NOS:409 and
408, respectively) was fused to human IL-4 (SEQ ID NO:119), the
latter being attached to the C-terminus of the heavy chains with an
intervening six amino acid linker, L6 (SGGGGS, SEQ ID NO:132). In
addition to this J110-HC-L6-IL4 IgG1 protein (composed of SEQ ID
NOS:304 (heavy chain) and 300 (light chain)), a variant of this
with a single substitution in the IL-4 component, J110-HC-L6-IL-4
(R88Q) IgG1 (composed of SEQ ID NOS:306 (heavy chain) and 300
(light chain)) was made. The R88Q mutation in IL-4 has been
reported to reduce its potency in vitro (Kruse, EMBO Journal vol.
12 no. 13 pp. 5121-5129, 1993).
Methods:
[0377] The "off-target (HB-IL4) assay" was performed largely as
described by the manufacturer of the HEK-Blue IL4/IL13 cell line.
HEK-Blue.TM. IL-4/IL-13 Cells are specifically designed to monitor
the activation of the STAT6 pathway, which is induced by IL-4. The
cells were generated by introducing the human STAT6 gene into
HEK293 cells to obtain a fully active STAT6 signaling pathway. The
HEK-Blue.TM. IL-4/IL-13 Cells stably express a reporter gene,
secreted embryonic alkaline phosphatase (SEAP), under the control
of the IFN.beta. minimal promoter fused to four STAT6 binding
sites. Activation of the STAT6 pathway in HEK-Blue.TM. IL-4/IL-13
cells induces the expression of the SEAP reporter gene. SEAP is
then secreted into the media and can be quantitated using the
colorimetric reagent QUANTI-Blue.TM.. Briefly, HEK-Blue IL4/IL13
cells (Invivogen, San Diego Calif. cat# hkb-stat6) were thawed and
cultured in DMEM media (Mediatech, Manassas Va., cat#10-013-CV)+10%
FBS (Hyclone, Logan Utah, cat# SH30070.03) that had been heat
inactivated (HI FBS). After one passage, 10 .mu.g/ml blasticidin
(Invivogen cat# ant-bl-1) and 100 micrograms/ml Zeocin (Invivogen
cat# ant-zn-1) were added to the culture medium. After one more
passage, cells were allowed to reach 60-80% confluence and then
lifted with Cell Stripper (Mediatech, cat#25-056-Cl). Cells were
washed twice in DMEM+HI FBS and counted. Cells were adjusted to
2.8.times.10.sup.5 viable cells/ml in DMEM+HI FBS and 180 .mu.l was
aliquoted per well into a flat bottom 96 well tissue culture plate
(hereafter, the "experimental plate"). Then, 20 .mu.l of IL-4 or
fusion protein construct, diluted into DMEM+HI FBS, was added per
well. The plate was incubated at 37.degree. C. 5% CO.sub.2 for
16-24 hours. QUANTI-Blue (Invivogen, cat# rep-qbl) was prepared
according to the manufacturer's directions. QUANTI-Blue (160 .mu.l)
was aliquoted into each well of a flat bottom plate (hereafter, the
"assay plate"). Then, 40 .mu.l supernatant per well from the
experimental plate was transferred to assay plate. Assay plate was
then incubated at 37.degree. C. for 1-3 hours. Assay plate
absorbance at 630 nm was read on a model 1420-41 Victor 3V
Multilabel Counter from Perkin-Elmer. Data was analyzed using Graph
Pad Prism.
[0378] The "on target (Th1 diversion) assay" was designed to
monitor the percentage of CD4.sup.+ T cells that were of Th1
phenotype, as defined by their expression of IFN-.gamma.. Th1
diversion is thereby quantified by a decrease in
IFN-.gamma.-positive CD4 T cells. The assay was performed as
follows: "Loaded" Dynabeads (M450 Epoxy beads, Invitrogen Dynal,
Oslo, Norway cat#140.11) were made as described by the manufacturer
with 1.0 .mu.g/10.sup.7 beads anti-human CD3 epsilon antibody
(R&D Systems, Minneapolis Minn., cat# MAB100), 1.0
.mu.g/10.sup.7 beads anti-human CD28 antibody (R&D Systems,
cat# MAB342) and 3 .mu.g/10.sup.7 beads human IgG (R&D Systems,
cat#1-001-A). PBMCs were obtained from the Stanford Blood Center;
Palo Alto Calif. Naive CD4.sup.+ T cells were purified from
Leukocyte Reduction System (LRS) cones using the naive CD4.sup.+
kit (Miltenyi Biotech cat#130-094-131) according to the
manufacturer's directions. A total of 4.0.times.10.sup.5 purified
naive CD4.sup.+ T-cells were aliquoted to each well of a 24 well
tissue culture plate (hereafter, the "experimental plate") in 1.3
ml in RPMI 1640 (Mediatech, cat#10-040-CV)+10% HI FBS+100 units/ml
IL-2 (Peprotech, cat#200-02), hereafter referred to as Media-Q.
Then, 4.0.times.10.sup.5 "loaded" Dynabeads were added per well.
IL-12 (Peprotech, cat#200-12) was diluted into Media-Q and 100
.mu.l was added to appropriate wells, giving a final concentration
of 10 ng/ml. Fusion protein constructs or IL-4 were diluted into
Media-Q and 100 was added to the appropriate wells. Media-Q was
added to appropriate wells to bring the total volume of each well
to 1.5 ml. The experimental plate was incubated at 37.degree. C.
with 5% CO.sub.2 for five days. On the morning of the fifth day,
Phorbol myristate acetate (PMA) was added to all wells at a final
concentration of 50 ng/ml and ionomycin was also added to all wells
at a final concentration of 1.0 Brefeldin A was added to a final
concentration of 1.0 .mu.g/ml of culture. The experimental plate
was incubated at 37.degree. C. with 5% CO.sub.2 for a minimum of
four hours. Approximately 1/3 of the volume of each well of the
experimental plate was then recovered and subjected to preparation
for Intra-cellular Flow Cytometry according to the instructions
supplied with the abovementioned Kit and utilizing the reagents
supplied with the kit. The cells were stained for intra-cellular
interferon-gamma with an anti human interferon-gamma antibody
conjugated to AF647 (eBiosciences.com, cat#51-7319-42). The samples
were analyzed by flow cytometry on a Becton Dickinson FACSort using
Cell Quest software. Acquired samples were analyzed using FloJo
Software and data were graphed using Graph Pad Prism software.
Results:
[0379] In the absence of antibody-based targeting, as measured by
the "off-target (HB-IL4) assay," IL4 showed an EC50 of 1.26 pM
(FIG. 44; Table 35). The attachment of an IgG1 to IL4 (e.g. the
construct J110-HC-L6-IL4 IgG1, in which the wild type human IL-4
sequence was attached to the C-terminus of the chimeric J110
antibody that recognizes PD-1, with an intervening linker L6)
reduced the potency by 5.46-fold (6.88/1.26=5.46). By introducing
the R88Q point mutation into the IL-4 portion of this construct,
the potency was further reduced to 35,600-fold (44,800/1.26=35,555)
below free IL-4. A second antibody-IL-4 (R88Q) fusion protein
construct (Isotype-HC-L6-IL4 (R88Q) IgG1, composed of SEQ ID
NOS:358 (heavy chain) and 344 (light chain)) showed similar
potency. The isotype antibody used in this experiment was 2D12.
TABLE-US-00038 TABLE 35 Antigen EC50 On EC50 EC50 Off EC50
Specificity Target (pM) IL4/EC50 TA Target IL4/EC50 TA Index Test
Article Th1 (Th1 (pM) HB- (Off Target; Column 3/ (TA) Diversion
Diversion) IL4 HB-IL4) Column 5 IL4 11.4 1.00 1.26 1.00 1.00
J110-HC-L6- 31.8 0.358 6.88 0.183 1.96 IL4 IgG1 J110-HC-L6- 46.1
0.247 44,800 2.81 .times. 10.sup.-5 8,790 IL4 (R88Q) IgG1
Isotype-HC- >1000 ND 19,200 6.56 .times. 10.sup.-5 ND L6-IL4
(R88Q) IgG1
[0380] The "on target (Th1 diversion) assay" results are shown in
FIG. 45. Activation of the naive (Th0) CD4 cells induces PD-1
expression, so that the anti-PD1-IL-4 fusion protein constructs may
target the IL-4 to them. In this assay, free, wild type IL4 shows
an EC50 of 11.4 pM. Remarkably, the anti-PD1-attenuated IL-4 fusion
protein construct (J110-HC-L6-IL4 (R88Q) IgG1), which was
35,600-fold less potent than free, wild type IL-4 in the
"off-target (HB-IL4) assay," was almost as potent as IL-4 in this
on-target assay (1/4.sup.th as potent; 11.4/46.1=0.25). The
non-attenuated, PD-1 targeted fusion protein construct
(J110-HC-L6-IL4 IgG1) was only slightly more potent than the
attenuated form (1.45.times. more potent; 46.1/31.8=1.45). The
non-targeted, attenuated IL-4 fusion protein construct
(Isotype-HC-L6-IL4 R88Q) IgG1, was significantly less potent than
the targeted attenuated fusion protein construct, but its potency
was too low to accurately determine an EC50 in this experiment.
Interleukin-6 (IL-6)
[0381] IL-6 (SEQ ID NO:123) has numerous activities on different
cell types and it may be advantageous to exploit some of these
activities at the expense of others. For example, by targeting
newly activated CD4.sup.+ T cells (via attachment to an anti-PD1
antibody, as in the example above with IL-4 targeting, for
example), one may shift the T helper cell population away from the
Treg pathway and in favor of the Th17 pathway. This could be
advantageous to a cancer patient.
Methods:
[0382] The "IL-6 bioassay" was performed using the HEK-Blue.TM.
IL-6 cells (Invivogen, cat# hkb-il6), an engineered reporter cell
line that monitors the activation of the JAK-STAT pathway by IL-6.
These cells were generated by introducing the human IL-6R gene into
HEK293 cells. In addition, cells were further transfected with a
reporter gene expressing SEAP under the control of the IFN.beta.
minimal promoter fused to four STAT3 binding sites. In these cells,
IL-6 stimulates the activation of STAT3 and leads to the secretion
of SEAP. SEAP is then monitored when using the SEAP detection
medium QUANTI-Blue.TM.. The assay was run essentially according to
the manufacturer's (Invivogen) instructions. Briefly, HEK-Blue IL6
cells were thawed and cultured in DMEM (Mediatech, Manassas Va.,
cat#10-013-CV)+10% FBS (Hyclone, Logan Utah, cat# SH30070.03) that
had been heat inactivated (HI FBS). After one passage, 200 .mu.g/ml
HygroGold, (Invivogen cat# ant-hg-1) and 100 .mu.g/ml Zeocin,
(Invivogen cat# ant-zn-1) was added to the culture medium. After
one more passage, cells were allowed to reach 60-80% confluence and
then lifted with Cell Stripper (Mediatech, cat#25-056-Ck). Cells
were then washed twice in DMEM+HI FBS and counted. Cells were
adjusted to 2.8.times.10.sup.5 viable cells/ml in DMEM+HI FBS and
180 ul was aliquoted per well into a flat bottom 96 well tissue
culture plate (hereafter, the "experimental plate"). Then, 20 .mu.l
of IL-6 or fusion protein construct, diluted into DMEM+HI FBS, was
added per well. The plate was incubated at 37.degree. C. with 5%
CO.sub.2 for 16-24 hours. QUANTI-Blue (Invivogen, cat# rep-qbl),
prepared according to the manufacturer's instructions, was then
aliquoted (160 .mu.l per well) into each well of a flat bottom
plate (hereafter, the "assay plate"). Then, 40 .mu.l supernatant
per well from the experimental plate was transferred to the wells
of the assay plate. The assay plate was incubated at 37.degree. C.
for 1-3 hours. Assay plate absorbance at 630 nm was read on a model
1420-41 Victor 3V Multilabel Counter from Perkin-Elmer. Data was
analyzed using Graph Pad Prism.
[0383] In order to test whether IL-6 can be attenuated and
targeted, such that a high Antigen Specificity Index (ASI) may be
achieved, IL-6 carrying a 16-mer linker (L16, SGGGGSGGGGSGGGGS, SEQ
ID NO:133) at the N-terminus was fused to an antibody targeting
class I MHC, using the HB95 antibody (which binds to human class I
MHC antigen, as described above) vs. an isotype control antibody,
2D12 (also described above). The non-targeting, isotype control
fusion protein construct, 2D12-HC-L16-IL6 IgG1 (composed of SEQ ID
NOS:360 (heavy chain) and 344 (light chain)), was compared to free
IL-6 in the "IL-6 bioassay" described above (FIG. 46). The antibody
fusion showed about 10-fold lower potency than free IL-6
(10.9/1.04=10.5). By introducing the R179E mutation (known to
reduce the potency of IL-6; Kalai, Blood 89(4):1319-33, 1997)) into
this fusion protein construct, the resulting construct
(2D12-HC-L16-IL6(R179E) IgG1, composed of SEQ ID NOS:362 (heavy
chain) and 344 (light chain)) was further attenuated, showing a
potency 79,400-fold lower than free, wild type IL-6
(82,600/1.04=79,400). By contrast, when the attenuated IL-6 was
attached to an antibody (HB95) that binds to an antigen (class I
MHC) on the HEK-Blue.TM. IL-6 cells (HB95-HC-L16-IL-6(R179E) IgG1,
composed of SEQ ID NOS:324 (heavy chain) and 312 (light chain)),
the potency was increased compared to the non-targeted
antibody-attenuated IL-6 fusion protein construct by 953-fold
(82,600/86.7=953). This potency is only 6.99-fold lower than that
of the targeted, wild type IL-6 fusion protein construct
(HB95-HC-L16-IL6 IgG1, composed of SEQ ID NOS:322 (heavy chain) and
312 (light chain); 86.7/12.4=6.99). In other words, in the absence
of antibody-antigen targeting and in the context of antibody-IL-6
fusion protein constructs, the R179E mutation reduces the IL-6
potency by 7,580-fold (82,600/10.9=7,580) compared to the mere
6.99-fold in the presence of targeting.
In Vivo Studies of Antibody-Targeted Attenuated IFN.alpha.
[0384] To confirm that the antibody-attenuated ligand fusion
protein constructs of the present invention were active in vivo
several experiments, using constructs consisting of antibodies to
CD38, which is expressed on the surface of multiple myeloma cells
and attenuated versions of IFN.alpha.2b, were performed. In most
studies this was compared to non-targeted control fusion protein
constructs referred to below as "isotype control". The variable
regions for the isotype control antibodies were derived from the
antibody 2D12 which was raised against the yellow fever virus
(Shlesinger, Virology 125: 8-17, 1983).
[0385] In the first experiment, a xenograft model in which the
multiple myeloma cell line NCI-H929 (ATCC CRL-9068, Gazdar, Blood
67: 1542-1549, 1986) is grown subcutaneously in immunocompromised
(SCID) mice was used.
Methods:
[0386] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When average tumor size reached 120-150
mm.sup.3, mice were grouped into 5 cohorts of 10 mice each and
treatment began at time zero (TO). All treatments were given by
intraperitoneal injection, (i.p.) twice weekly for 5 weeks
(indicated by bar under graph). All compounds were dosed at 200
.mu.g/dose (approximately 10 mg/kg) except for Interferon-.alpha..
IFN.alpha.2b (Intron A.RTM., Schering Corp., Merck, Whitehouse
Station, N.J.) was given at 2 million units/dose. Tumor volume was
measured twice weekly by caliper measurement. Endpoint was tumor
volume of 2,000 mm.sup.3.
Results:
[0387] Results are shown in FIG. 47. Treatment of this multiple
myeloma subcutaneous (s.c.) solid tumor with interferon-.alpha.
(closed diamonds) slightly delayed tumor growth in these mice
compared to vehicle (P<0.05, closed circles). Treatment with
naked anti-CD38 antibody (G005 IgG1, composed of SEQ ID NOS:135
(heavy chain) and 134 (light chain)), (closed squares) had no
significant effect on tumor growth compared to vehicle. All mice in
these two groups reached endpoint (2,000 mm.sup.3) by day 30. The
non-targeted isotype control-attenuated IFN.alpha. fusion protein
construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG1, composed of SEQ
ID NOS:348 (heavy chain) and 344 (light chain) (open inverted
triangles) did show significant activity in delaying tumor growth,
presumably due to the long half-life of the antibody-IFN.alpha.
fusion protein construct and resulting increased systemic exposure.
The CD38-targeted attenuated IFN.alpha. fusion protein construct
(G005-HC-L6-IFN.alpha. (A145G) IgG1, composed of SEQ ID NOS:144
(heavy chain) and 134 (light chain)), by contrast, showed dramatic
anti-tumor activity compared to the non-targeted fusion protein
construct (P<0.0001.) or the other test substances. The targeted
anti-CD38-attenuated IFN.alpha. fusion protein construct completely
resolved tumors in all (10/10) mice to undetectable levels by day
22 with no recurrence throughout the duration of the study.
[0388] The anti-CD38-attenuated IFN.alpha. fusion protein construct
(G005-HC-L6-IFN.alpha. (A145G) IgG1) was tested in a systemic
multiple myeloma model based on the cell line MM1S (Crown
Bioscience Inc., Santa Clara; Greenstein, Exp Hematol. April;
31(4):271-82,2003).
Methods:
[0389] Six to 8 week old NOD-SCID mice were injected intravenously
with 1.times.10.sup.7MM1S tumor cells in 0.1 ml phosphate buffered
saline (PBS) 24 hours after irradiation with 200 rad (.sup.60Co).
Mice were grouped into 4 cohorts of 10 mice each at time zero and
treatments began 7 days later. All treatments were given i.p. twice
weekly for 9 weeks. All compounds were dosed at 200 .mu.g/dose
(approximately 10 mg/kg) except Interferon-.alpha. (given at 2
million units/dose). Body weights and overall health were monitored
twice weekly and survival was the endpoint.
Results:
[0390] Results are shown in FIG. 48. Treatment of this systemic
multiple myeloma tumor with interferon-.alpha. (Intron A) alone
increased median survival time (MST) by 18 days compared to vehicle
(MST 74 vs 56, respectively.) Treatment with naked anti-CD38
antibody (G005) only slightly increased survival (MST 62 days).
None of the mice in the targeted anti-CD38-attenuated IFN.alpha.
(G005-HC-L6-IFN.alpha. (A145G) IgG1) treated cohort showed signs of
disease during entire study. All (10/10) mice appeared healthy at
termination.
[0391] An in vivo study using a third model of cancer, based on the
Burkitt's lymphoma cell line Daudi (ATCC CCL-213, Klein, Cancer
Res. 28: 1300-1310, 1968) was performed. Daudi cells are CD38+.
Methods:
[0392] Six to eight week old NOD-SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 Daudi Burkitt's
Lymphoma tumor cells in 50% Matrigel one day after irradiation with
200 rad (.sup.60Co). When mean tumor size reached 169 mm.sup.3 (Day
20), mice were grouped into 5 cohorts of 10 mice each and treatment
began. All treatments were given i.p. twice weekly for 4 weeks. All
compounds were dosed at 200 .mu.g/dose (approximately 10 mg/kg)
except Interferon-.alpha., which was given at 2 million units
(MIU)/dose. Tumor volume was measured twice weekly by caliper
measurement. Endpoint was tumor volume of 2,000 mm.sup.3.
Results:
[0393] Results are shown in FIG. 49. Treatment of this Burkitt's
lymphoma s.c. tumor with the naked anti-CD38 antibody (closed
square) did not significantly delay tumor growth in these mice
compared to vehicle (closed circles). The IFN.alpha. treatment did
result in a significant delay in tumor growth compared to vehicle
(5.5 days) however this group reached the 2,000 mm.sup.3 endpoint
by day 40. The non-targeted isotype control fusion protein
construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG1; open inverted
triangles) showed significant activity in delaying tumor growth but
this group reached the 2,000 mm.sup.3 endpoint on day 57. As
observed in the H929 model (above), this non-targeted activity is
most likely due to the extended half-life of the interferon,
thereby increasing exposure of the tumor to the cytokine. The
targeted anti-CD38-attenuated interferon fusion protein construct
(closed triangles) dramatically resolved tumors such that none of
the mice had palpable tumors by day 30. Some of the mice in this
group, however, did show re-growth of tumors after discontinuation
of treatment. Further analysis of this data is presented in Table
36.
TABLE-US-00039 TABLE 36 Median Tumor Size (mm3).sup.a T/C.sup.b
Treatment at Day 37 (%) P value.sup.c Vehicle 3034 +/- 340 -- --
Anti-CD38 (G005) IgG1 2443 +/- 196 81 0.575 G005-HC-L6-IFN.alpha.
(145G) IgG1 0 0 <0.0001 Isotype-HC-L6-IFN.alpha. (145G) IgG1 15
0.5 <0.0001 IFN.alpha. 1440 +/- 154 47 0.007 .sup.aMean +/- SEM
.sup.bRatio of tumor size for treatment group divided by tumor size
for vehicle group at day 37 .sup.cVs. vehicle control at day 37
[0394] This xenograft experiment shows that the CD38-targeted
attenuated IFN.alpha. fusion protein constructs may be effective in
treating lymphomas in addition to multiple myelomas.
[0395] The effect of different doses of the anti-CD38-attenuated
IFN.alpha. fusion protein construct, were compared to the
non-CD38-targeted fusion protein construct, on myeloma tumor
growth. For these comparisons, the NCI-H929 s.c. multiple myeloma
model was used.
Methods:
[0396] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When mean tumor sizes reached 120-150
mm.sup.3, mice were grouped into 9 cohorts of 10 mice each and
treatment began (time zero). All treatments were given i.p. twice
weekly for 5 weeks. Two compounds, targeted anti-CD38-attenuated
interferon (closed grey symbols) and non-targeted isotype
control-interferon (open symbols), were compared in this study at
different doses (see legend for doses). Tumor volume was measured
twice weekly by caliper measurement. Endpoint was tumor volume of
2,000 mm.sup.3.
Results:
[0397] Results are shown in FIG. 50. The dose titration of
anti-CD38-targeted attenuated IFN.alpha. fusion protein construct
(G005-HC-L6-IFN.alpha. (A145G) IgG1) demonstrated significant
efficacy at all doses of compound tested, even at 0.01 mg/kg.
Complete tumor elimination was observed in 10/10 mice only at the
highest (10 mg/kg) dose. By contrast, the isotype
control-attenuated IFN compound (Isotype-HC-L6-IFN.alpha. (A145G)
IgG1) showed significant activity only at the highest dose (10
mg/kg). The 0.01 mg/kg dose of the CD38 targeted, attenuated IFN
showed similar anti-tumor activity to the isotype control
attenuated IFN fusion protein construct at a 1,000-fold higher dose
(10 mg/kg), emphasizing the importance of the CD38-targeting.
[0398] The next example shows that antibodies of the present
invention also include those of the IgG4 isotype.
Methods:
[0399] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When average tumor size reached 120-150
mm.sup.3, mice were grouped into 5 cohorts of 10 mice each and
treatment began (time zero). All treatments were given i.p. twice
weekly for 5 weeks. All compounds were dosed at 70 .mu.g/dose
(approximately 3.5 mg/kg). Tumor volume was measured twice weekly
by caliper measurement. Endpoint was 2,000 mm.sup.3.
Results:
[0400] Results are shown in FIG. 51. This study compared the
activity of the targeted vs. non-targeted fusion protein constructs
in two different isotype formats; IgG1 isotype
(G005-HC-L6-IFN.alpha. (A145G) IgG1 (targeted, closed squares) and
Isotype-HC-L6-IFN.alpha. (A145G) IgG1 (non-targeted, open squares))
and IgG4 isotype (G005-HC-L6-IFN.alpha. (A145G) IgG4, composed of
SEQ ID NOS:148 (heavy chain) and 134 (light chain) (targeted,
closed diamonds) and Isotype-HC-L6-IFN.alpha. (A145G) IgG4,
composed of SEQ ID NOS:350 (heavy chain) and 344 (light chain)
(non-targeted, open diamonds)) were compared. It is important to
note that the mice in this study were treated at a lower dose than
in previous studies where we observed 100% tumor elimination. The
tumor volumes indicate that, surprisingly, the IgG4 format is more
potent than the IgG1 format in this model. Since human IgG1
antibodies have greater effector function than IgG4 antibodies
(Hooper, Ann Clin Lab Sci.; 8:201, 1978; Ward, Ther Immunol, 2:77,
1995.), it would have been expected that the IgG1 format would have
been at least as effective, if not more so, than the IgG4 format.
At the end of study, 8/10 mice in the CD38 targeted, attenuated
IFN, IgG4 treated group (closed diamonds) were tumor free whereas
only 3/10 were tumor-free in the IgG1 format counterpart (closed
squares).
[0401] The next example extends the observation of in vivo efficacy
of an antibody-targeted IFN to a second mutated form of IFN.alpha.
in which A145 has been mutated to aspartic acid (D). In addition,
the experiment below utilizes a different CD38 antibody, i.e. one
based on the variable regions of human antibody clone X355/02; (SEQ
ID NOS:391 (VH) and 390 (VX)). A third difference between this
construct and the one presented in the preceding in vivo
experiments is that the linker is removed (referred to as "L0"),
i.e. the mutated IFN.alpha. is fused directly to the C terminus of
the antibody heavy chain.
Methods:
[0402] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When average tumor size reached 120-150
mm.sup.3, mice were grouped into 3 cohorts of 10 mice each and then
treatment began (time zero). All treatments were given i.p. twice
weekly for 5 weeks. All compounds were dosed at 60 .mu.g/dose
(approximately 3 mg/kg). Tumor volume was measured twice weekly by
caliper measurement. Endpoint was tumor volume of 2,000
mm.sup.3.
Results:
[0403] Results are shown in FIG. 52. This anti-CD38-attenuated
IFN.alpha. fusion protein construct (X355/02-HC-L0-IFN.alpha.
(A145D) IgG4, composed of SEQ ID NOS:232 (heavy chain) and 226
(light chain)) was also very effective in tumor elimination,
showing that the ability of anti-CD38-attenuated IFN.alpha. fusion
protein constructs to effectively treat human myeloma in an in vivo
model is not restricted to a single variable domain, IFN.alpha.
mutation or linker between the antibody and the IFN. The isotype
control fusion protein construct showed significantly less
anti-myeloma activity, consistent with the CD38-based
targeting.
[0404] The next example shows that an anti-CD38 antibody-attenuated
IFN.alpha. fusion protein construct is more effective than standard
drugs used to treat multiple myeloma in the same xenograft model
described above.
Methods:
[0405] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When mean tumor sizes reached 120-150
mm.sup.3, mice were grouped into cohorts of 10 mice each and
treatment began (time zero). Treatments were administered at doses
and regimens described in legend. Tumor volume was measured twice
weekly by caliper measurement. Endpoint was 2,000 mm.sup.3.
Results:
[0406] Results are shown in FIG. 53. In this study the activity of
the anti-CD38 targeted fusion protein construct
(G005-HC-L0-IFN.alpha. (A145D)IgG4, composed of SEQ ID NOS:180
(heavy chain) and 134 (light chain)) was compared with standard
therapies Bortezomib (Velcade), Melphalan (Alkeran), and
Dexamethasone. Of the anti-CD38 targeted, attenuated interferon
group, 8/10 were tumor free at day 60 (closed triangles), whereas
all mice in the other groups had reached endpoint by day 50.
[0407] The next example shows that an anti-CD38-attenuated IFN
fusion protein construct can completely eliminate established human
multiple myeloma tumors in a mouse model, even when the fusion
protein construct is given as a single dose.
Methods:
[0408] Eight to 12 week old CB.17 SCID mice were injected
subcutaneously in the flank with 1.times.10.sup.7 NCI-H929 tumor
cells in 50% Matrigel. When mean tumor sizes reached 120-150
mm.sup.3 mice were grouped into cohorts of 10 mice each and then
treatment began (TO). Treatments with the anti-CD38
antibody-attenuated Interferon fusion protein construct were
administered according to following regimens: single dose on day 0
(closed triangles), two doses (on day 0 and day 3; closed squares),
4 doses (on days 0, 3, 8, and 11; closed diamonds) and 6 doses (on
days 0, 3, 8, 11, 15 and 18; closed black circles). One cohort
received 6 doses of the isotype control-attenuated interferon
fusion protein construct on days 0, 3, 8, 11, 15 and 18 (open
squares). The vehicle treatment group is shown in grey filled
circles. Tumor volume was measured twice weekly by caliper
measurement. Endpoint was 2,000 mm.sup.3.
Results:
[0409] Results are shown in FIG. 54. This study surprisingly showed
that a single dose of the G005-HC-L6-IFN.alpha. (A145G) IgG4 fusion
protein construct was sufficient to eliminate established tumors in
all 10/10 mice by day 15; furthermore, by day 60, no tumors had
re-grown in any of the mice in this single dose group. This was
true of all 4 dosing regimens with the targeted attenuated
interferon. The isotype control group was only tested at the 6 dose
regimen and showed considerably less activity. That a single dose
of a compound can effectively cure animals of established multiple
myeloma tumors is unprecedented and extremely surprising since
anti-tumor therapies are typically dosed multiple times in order to
observe efficacy.
[0410] The next example demonstrates that even very large tumors
can be eliminated by treatment with an anti-CD38-attenuated IFN
fusion protein construct.
Methods:
[0411] One cohort (n=9) from the immediately preceding study was
not treated until the mean tumor volume reached 730 mm.sup.3. This
cohort then received 6 doses of anti-CD38 targeted, attenuated
interferon on days 12, 15, 19, 22, 26 and 29 (arrows).
Results:
[0412] Results are shown in FIG. 55. 8/9 mice in this cohort showed
complete tumor elimination by day 30 and no tumors had re-appeared
in any of these mice by end of the study. Three of these mice had
starting tumors >1000 mm.sup.3. The only mouse that died from
the myeloma was one which had a tumor volume of 1,800 mm.sup.3 at
the start of treatment; it reached the 2,000 mm.sup.3 enpoint on
the following day. This result is very surprising and no other
compound has been reported to eliminate myeloma tumors of this size
in any animal model.
[0413] It was shown that in the in vitro experiments above (Table
27) that the G005-HC-L6-IFN.alpha. (A145G) IgG4 fusion protein
construct has about 25,000-fold lower potency than free, wild-type
IFN.alpha.2b under conditions where the antibody does not target
the attenuated IFN.alpha. to the cells being tested (off target
assay). The following experiments aimed to determine if the fusion
protein construct also showed dramatic attenuation of IFN activity
in an ex vivo assay of IFN activity that is relevant to the
toxicity of IFN.alpha.. This effect of IFN.alpha. on hematopoiesis
can be measured ex vivo by determining the effect of IFN.alpha. on
the number of colony forming units derived from primary human bone
marrow mononuclear cells. The IFN.alpha. vs. the
antibody-attenuated IFN.alpha. fusion protein constructs were
compared in terms of their effect on colony formation.
Methods:
[0414] Frozen normal human bone marrow mononuclear cells (AllCells,
Inc., Emeryville, Calif.) from 3 donors were thawed in RPMI-1640
medium plus 10% fetal bovine serum (FBS) (complete medium) and
washed with same medium two times. After washing, cells were kept
in this medium at 1.75.times.10.sup.6 cells/ml. Cell suspensions
were diluted with MethoCult H4434 Classic medium (Stem Cell
Technologies, Cat#04434) to a final cell concentration of
0.7.times.10.sup.5 cells/ml. Cells were then mixed very well and 3
ml of this mixture was aliquoted into each tube.
[0415] Intron A (Schering Corp. Merck, N.J.) and fusion protein
constructs (G005-HC-L0-IFN.alpha. (145D) IgG4 and
Isotype-HC-L0-IFN.alpha. (145D) IgG4) were diluted in tenfold
serial dilutions in complete medium and 150 .mu.l of each dilution
was added to tubes containing the 3 ml of the bone marrow cells in
the Methocult H4434 medium. Mixtures were plated at 1.1 ml per 35
mm tissue culture dish (Stem Cell Technologies, cat#27115). Plates
were then incubated in a well-humidified incubator at 37.degree. C.
with 5% CO.sub.2 for two weeks. Colonies were counted on a
microscope using a gridded scoring dish (Stem Cell Technologies,
Cat#27500) and the number of colonies/plate was recorded. Percent
colony recovery for a given test substance was calculated by
dividing the number of colonies per plate by the number of colonies
in the plates with no added test substance. A total of three human
bone marrow MNC were tested using this method.
Results:
[0416] The results are shown in FIG. 56. The data indicate that
both the targeted (anti-CD38, G005) and the non-targeted (isotype;
2D12) attenuated interferon fusion protein constructs had similar
activity, indicating that the CD38 expression observed on normal
bone marrow cells is not likely expressed on the colony forming
cells since very little inhibition of colony formation was observed
with the targeted treatment. Both fusion protein constructs had
approximately 10,000.times. fold less activity in inhibiting colony
formation than wild type, free IFN.alpha., thus confirming that the
A145D mutation attenuates the IFN activity of the
antibody-IFN.alpha. fusion protein constructs and suggesting that
such attenuated IFN-antibody fusion protein constructs will have a
superior safety profile compared to IFN.alpha. itself.
[0417] Another activity of IFN.alpha. that can be measured ex vivo
is the stimulation of cytokine and chemokine secretion. Normal
human PBMCs were stimulated with various concentrations of
IFN.alpha. vs the antibody-attenuated IFN.alpha. fusion protein
construct Isotype-HC-L6-IFN.alpha. (A145G) IgG1 (based on the 2D12
antibody), and measured the resulting cytokine production.
Methods:
[0418] Normal human peripheral blood mononuclear cells (PBMC) from
four normal donors were washed with Xvivo-15 medium (Lonza,
Cat#04-418Q) and resuspended in the same medium at a cell density
of 1.times.10.sup.6 cells/ml. The cells were then incubated with
human IgG at 4 mg/ml and incubated at 37.degree. C. for 30 min to
block any nonspecific IgG binding. Without washing, 250 .mu.l
aliquots of cells were then added to wells of 24-well tissue
culture treated plates. To these wells were then added 250 .mu.l of
free IFN.alpha. or an IgG-attenuated IFN.alpha. fusion protein
construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG1; isotype antibody
is 2D12) at various concentrations. Plates were then incubated
overnight at 37.degree. C. in 5% CO.sub.2. The following day, the
plates were spun down and 200 .mu.l of supernatant was collected
from each well. Supernatants were kept frozen until analysis using
a Luminex cytokine assay.
[0419] Luminex Assay: Using the Premix 42-plex from Millipore
(Cat#MPXHCYTO60KPMX42) we were able to measure the level of human
cytokines produced by the PBMCs stimulated by the test substances.
The culture supernatants were incubated with the pre-mixed
polystyrene microbeads that were coated with anti-cytokine
antibodies according to the manufacturer's instructions. After
washing, the biotinylated detection antibody cocktail was
introduced to the bead-captured analyte. Finally the reaction
mixture was incubated with Streptavidin PE and the fluorescent
intensity of PE was measured on the Luminex analyzer. Results were
interpolated by the standard curve constructed based on the
controls provided in the kit.
Results:
[0420] Results are shown in FIG. 57. Four cytokines (IP-10, MCP-1,
MCP-3 and IL-1.alpha.) were consistently upregulated in response to
IFN.alpha. exposure. The antibody-attenuated IFN.alpha. fusion
protein construct (Isotype-HC-L6-IFN.alpha. (A145G) IgG1) showed
1,000-5,000-fold reduced potency compared to wild type IFN.alpha.
stimulation. This confirms that the mutation did result in a
significant attenuation of biological activity. Panel (a) shows the
dose-response curves for IP-10 (estimated at 1,000-fold
attenuation) and MCP-1 (estimated at about 5,000-fold attenuation);
panel (b) shows the dose-response curves for MCP-3 (estimated at
.about.2,500-fold attenuation) and IL1.alpha. (estimated at about
1,300-fold attenuation).
Sequence Tables
TABLE-US-00040 [0421] TABLE 37 Single polypeptide chain sequences
SEQ ID NO: Species Length Unit Gene Subtype Variant 1 human 166 aa
IFN .alpha.1b native 2 human 165 aa IFN .alpha.2a native 3 human
165 aa IFN .alpha.2b native 4 human 165 aa IFN .alpha.2b L15A 5
human 165 aa IFN .alpha.2b A19W 6 human 165 aa IFN .alpha.2b R22A 7
human 165 aa IFN .alpha.2b R23A 8 human 165 aa IFN .alpha.2b S25A 9
human 165 aa IFN .alpha.2b L26A 10 human 165 aa IFN .alpha.2b F27A
11 human 165 aa IFN .alpha.2b L30A 12 human 165 aa IFN .alpha.2b
L30V 13 human 165 aa IFN .alpha.2b K31A 14 human 165 aa IFN
.alpha.2b D32A 15 human 165 aa IFN .alpha.2b R33K 16 human 165 aa
IFN .alpha.2b R33A 17 human 165 aa IFN .alpha.2b R33Q 18 human 165
aa IFN .alpha.2b H34A 19 human 165 aa IFN .alpha.2b Q40A 20 human
165 aa IFN .alpha.2b D114R 21 human 165 aa IFN .alpha.2b L117A 22
human 165 aa IFN .alpha.2b R120A 23 human 165 aa IFN .alpha.2b
R120E 24 human 165 aa IFN .alpha.2b R125A 25 human 165 aa IFN
.alpha.2b R125E 26 human 165 aa IFN .alpha.2b K131A 27 human 165 aa
IFN .alpha.2b E132A 28 human 165 aa IFN .alpha.2b K133A 29 human
165 aa IFN .alpha.2b K134A 30 human 165 aa IFN .alpha.2b R144A 31
human 165 aa IFN .alpha.2b R144D 32 human 165 aa IFN .alpha.2b
R144E 33 human 165 aa IFN .alpha.2b R144G 34 human 165 aa IFN
.alpha.2b R144H 35 human 165 aa IFN .alpha.2b R144I 36 human 165 aa
IFN .alpha.2b R144K 37 human 165 aa IFN .alpha.2b R144L 38 human
165 aa IFN .alpha.2b R144N 39 human 165 aa IFN .alpha.2b R144Q 40
human 165 aa IFN .alpha.2b R144S 41 human 165 aa IFN .alpha.2b
R144T 42 human 165 aa IFN .alpha.2b R144V 43 human 165 aa IFN
.alpha.2b R144Y 44 human 165 aa IFN .alpha.2b A145D 45 human 165 aa
IFN .alpha.2b A145E 46 human 165 aa IFN .alpha.2b A145G 47 human
165 aa IFN .alpha.2b A145H 48 human 165 aa IFN .alpha.2b A145I 49
human 165 aa IFN .alpha.2b A145K 50 human 165 aa IFN .alpha.2b
A145L 51 human 165 aa IFN .alpha.2b A145M 52 human 165 aa IFN
.alpha.2b A145N 53 human 165 aa IFN .alpha.2b A145Q 54 human 165 aa
IFN .alpha.2b A145R 55 human 165 aa IFN .alpha.2b A145S 56 human
165 aa IFN .alpha.2b A145T 57 human 165 aa IFN .alpha.2b A145V 58
human 165 aa IFN .alpha.2b A145Y 59 human 165 aa IFN .alpha.2b
M148A 60 human 165 aa IFN .alpha.2b R149A 61 human 165 aa IFN
.alpha.2b S152A 62 human 165 aa IFN .alpha.2b L153A 63 human 165 aa
IFN .alpha.2b N156A 64 human 165 aa IFN .alpha.2b L30A + YNS 65
human 165 aa IFN .alpha.2b R33A + YNS 66 human 165 aa IFN .alpha.2b
M148A + YNS 67 human 165 aa IFN .alpha.2b L153A + YNS 68 human 165
aa IFN .alpha.2b R144A + YNS 69 human 165 aa IFN .alpha.2b N65A,
L80A, Y85A, Y89A 70 human 165 aa IFN .alpha.2b N65A, L80A, Y85A,
Y89A, D114A 71 human 165 aa IFN .alpha.2b N65A, L80A, Y85A, Y89A,
L117A 72 human 165 aa IFN .alpha.2b N65A, L80A, Y85A, Y89A, R120A
73 human 165 aa IFN .alpha.2b Y85A, Y89A, R120A 74 human 165 aa IFN
.alpha.2b D114A, R120A 75 human 165 aa IFN .alpha.2b L117A, R120A
76 human 165 aa IFN .alpha.2b L117A, R120A, K121A 77 human 165 aa
IFN .alpha.2b R120A, K121A 78 human 165 aa IFN .alpha.2b R120E,
K121E 79 human 160 aa IFN .alpha.2b .DELTA.[L161-E165] 80 human 166
aa IFN .alpha.4b native 81 human 166 aa IFN .alpha.5 native 82
human 166 aa IFN .alpha.6 native 83 human 166 aa IFN .alpha.7
native 84 human 166 aa IFN .alpha.8 native 85 human 166 aa IFN
.alpha.10 native 86 human 166 aa IFN .alpha.1a/13 native 87 human
166 aa IFN .alpha.14 native 88 human 166 aa IFN .alpha.16 native 89
human 166 aa IFN .alpha.17 native 90 human 166 aa IFN .alpha.21
native 91 human 166 aa IFN .beta.1(a) native 92 human 166 aa IFN
.beta.1(a) R27A 93 human 166 aa IFN .beta.1(a) R35T 94 human 166 aa
IFN .beta.1(a) E42K 95 human 166 aa IFN .beta.1(a) D54N 96 human
166 aa IFN .beta.1(a) M62I 97 human 166 aa IFN .beta.1(a) G78S 98
human 166 aa IFN .beta.1(a) K123A 99 human 166 aa IFN .beta.1(a)
C141Y 100 human 166 aa IFN .beta.1(a) A142T 101 human 166 aa IFN
.beta.1(a) E149K 102 human 166 aa IFN .beta.1(a) R152H 103 human
166 aa IFN .beta.1(b) C17S 104 human 166 aa IFN .beta.1(b) C17S,
R35A 105 human 166 aa IFN .beta.1(b) C17S, R147A 106 human 143 aa
IFN .gamma. native 107 human 143 aa IFN .gamma. S20I 108 human 143
aa IFN .gamma. S20C 109 human 143 aa IFN .gamma. D21K 110 human 143
aa IFN .gamma. V22D 111 human 143 aa IFN .gamma. A23Q 112 human 143
aa IFN .gamma. A23V 113 human 143 aa IFN .gamma. D24A 114 human 141
aa IFN .gamma. .DELTA.[A23, D24] 115 human 141 aa IFN .gamma.
.DELTA.[N25, G26] 116 human 122 aa IFN .gamma. .DELTA.[A123-Q143]
117 human 129 aa IFN .gamma. .DELTA.[K130-Q143] 118 human 132 aa
IFN .gamma. .DELTA.[K130, R131, L135-Q143] 119 human 129 aa IL-4
native 120 human 129 aa IL-4 E9K 121 human 129 aa IL-4 R88D 122
human 129 aa IL-4 R88Q 123 human 184 aa IL-6 native 124 human 184
aa IL-6 F74E 125 human 184 aa IL-6 F78E 126 human 184 aa IL-6 R179E
127 human 310 aa CD38 human tagged, ECD 128 cynomolgus 310 aa CD38
cynomolgus tagged, ECD 129 human 774 nucleotide CD38 human ECD, for
genetic (coding immunisation (DNA) strand) 130 human 258 aa CD38
human ECD, for genetic immunisation (translated) 131 human 300 aa
CD38 human native 132 synthetic 6 aa linker 6-mer 133 synthetic 16
aa linker 16-mer
TABLE-US-00041 TABLE 38 SEQ ID NOs related to proteins comprising 2
polypeptide chains SEQ ID NO: Protein Name Chain Species Length
Unit 134 G005 IgG1 LC aa human 214 aa 135 HC aa human 452 aa 136 LC
DNA human 642 nucleotide (coding strand) 137 HC DNA human 1356
nucleotide (coding strand) 134 G005-HC-L0-IFN.alpha.(R144A) IgG1 LC
aa human 214 aa 138 HC aa synthetic 617 aa 136 LC DNA human 642
nucleotide (coding strand) 139 HC DNA synthetic 1851 nucleotide
(coding strand) 134 G005-HC-L6-IFN.alpha.(R144A) IgG1 LC aa human
214 aa 140 HC aa synthetic 623 aa 136 LC DNA human 642 nucleotide
(coding strand) 141 HC DNA synthetic 1869 nucleotide (coding
strand) 134 G005-HC-L0-IFN.alpha.(A145G) IgG1 LC aa human 214 aa
142 HC aa synthetic 617 aa 136 LC DNA human 642 nucleotide (coding
strand) 143 HC DNA synthetic 1851 nucleotide (coding strand) 134
G005-HC-L6-IFN.alpha.(A145G) IgG1 LC aa human 214 aa 144 HC aa
synthetic 623 aa 136 LC DNA human 642 nucleotide (coding strand)
145 HC DNA synthetic 1869 nucleotide (coding strand) 134
G005-HC-L6-IFN.alpha.(R144A) IgG4 LC aa human 214 aa 146 HC aa
synthetic 620 aa 136 LC DNA human 642 nucleotide (coding strand)
147 HC DNA synthetic 1860 nucleotide (coding strand) 134
G005-HC-L6-IFN.alpha.(A145G) IgG4 LC aa human 214 aa 148 HC aa
synthetic 620 aa 136 LC DNA human 642 nucleotide (coding strand)
149 HC DNA synthetic 1860 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha. IgG4 LC aa human 214 aa 150 HC aa synthetic
614 aa 136 LC DNA human 642 nucleotide (coding strand) 151 HC DNA
synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144A) IgG4 LC aa human 214 aa 152 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
153 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144D) IgG4 LC aa human 214 aa 154 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
155 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144E) IgG4 LC aa human 214 aa 156 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
157 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144G) IgG4 LC aa human 214 aa 158 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
159 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144H) IgG4 LC aa human 214 aa 160 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
161 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144I) IgG4 LC aa human 214 aa 162 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
163 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144K) IgG4 LC aa human 214 aa 164 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
165 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144L) IgG4 LC aa human 214 aa 166 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
167 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144N) IgG4 LC aa human 214 aa 168 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
169 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144Q) IgG4 LC aa human 214 aa 170 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
171 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144S) IgG4 LC aa human 214 aa 172 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
173 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144T) IgG4 LC aa human 214 aa 174 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
175 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144V) IgG4 LC aa human 214 aa 176 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
177 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(R144Y) IgG4 LC aa human 214 aa 178 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
179 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145D) IgG4 LC aa human 214 aa 180 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
181 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145E) IgG4 LC aa human 214 aa 182 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
183 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145G) IgG4 LC aa human 214 aa 184 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
185 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145H) IgG4 LC aa human 214 aa 186 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
187 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145I) IgG4 LC aa human 214 aa 188 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
189 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145K) IgG4 LC aa human 214 aa 190 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
191 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145L) IgG4 LC aa human 214 aa 192 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
193 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145N) IgG4 LC aa human 214 aa 194 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
195 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145Q) IgG4 LC aa human 214 aa 196 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
197 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145R) IgG4 LC aa human 214 aa 198 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
199 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145S) IgG4 LC aa human 214 aa 200 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
201 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145T) IgG4 LC aa human 214 aa 202 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
203 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145V) IgG4 LC aa human 214 aa 204 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
205 HC DNA synthetic 1842 nucleotide (coding strand) 134
G005-HC-L0-IFN.alpha.(A145Y) IgG4 LC aa human 214 aa 206 HC aa
synthetic 614 aa 136 LC DNA human 642 nucleotide (coding strand)
207 HC DNA synthetic 1842 nucleotide (coding strand) 208
G005-LC-L6-IFN.alpha.(A145G) IgG1 LC aa synthetic 385 aa 135 HC aa
human 452 aa 209 LC DNA synthetic 1155 nucleotide (coding strand)
137 HC DNA human 1356 nucleotide (coding strand) 210
G005-LC-L0-IFN.alpha.(A145G) IgG1 LC aa synthetic 379 aa 135 HC aa
human 452 aa 211 LC DNA synthetic 1137 nucleotide (coding strand)
137 HC DNA human 1356 nucleotide (coding strand) 134
G005-HC-L0-IFN.beta. IgG4 LC aa human 214 aa 212 HC aa synthetic
615 aa 136 LC DNA human 642 nucleotide (coding strand) 213 HC DNA
synthetic 1845 nucleotide (coding strand) 134
G005-HC-L0-IFN.beta.(R35A) IgG4 LC aa human 214 aa 214 HC aa
synthetic 615 aa 136 LC DNA human 642 nucleotide (coding strand)
215 HC DNA synthetic 1845 nucleotide (coding strand) 134
G005-HC-L0-IFN.beta.(R147A) IgG4 LC aa human 214 aa 216 HC aa
synthetic 615 aa 136 LC DNA human 642 nucleotide (coding
strand)
217 HC DNA synthetic 1845 nucleotide (coding strand) 218 MORAB03080
IgG1 LC aa human 212 aa 219 HC aa human 452 aa 220 LC DNA human 636
nucleotide (coding strand) 221 HC DNA human 1356 nucleotide (coding
strand) 222 hu38SB19 (SAR650984) IgG1 LC aa synthetic 214 aa 223 HC
aa synthetic 450 aa 224 LC DNA synthetic 642 nucleotide (coding
strand) 225 HC DNA synthetic 1350 nucleotide (coding strand) 226
X355/02 IgG1 LC aa human 222 aa 227 HC aa human 451 aa 228 LC DNA
human 666 nucleotide (coding strand) 229 HC DNA human 1353
nucleotide (coding strand) 226 X355/02-HC-L0-IFN.alpha.(R144A) IgG4
LC aa human 222 aa 230 HC aa synthetic 613 aa 228 LC DNA human 666
nucleotide (coding strand) 231 HC DNA synthetic 1839 nucleotide
(coding strand) 226 X355/02-HC-L0-IFN.alpha.(A145D) IgG4 LC aa
human 222 aa 232 HC aa synthetic 613 aa 228 LC DNA human 666
nucleotide (coding strand) 233 HC DNA synthetic 1839 nucleotide
(coding strand) 234 X355/07 IgG LC aa human 215 aa 235 HC aa human
448 aa 236 LC DNA human 645 nucleotide (coding strand) 237 HC DNA
human 1344 nucleotide (coding strand) 234
X355/07-HC-L0-IFN.alpha.(R144A) IgG4 LC aa human 215 aa 238 HC aa
synthetic 610 aa 236 LC DNA human 645 nucleotide (coding strand)
239 HC DNA synthetic 1830 nucleotide (coding strand) 234
X355/07-HC-L0-IFN.alpha.(A145D) IgG4 LC aa human 215 aa 240 HC aa
synthetic 610 aa 236 LC DNA human 645 nucleotide (coding strand)
241 HC DNA synthetic 1830 nucleotide (coding strand) 242 X910/12
IgG1 LC aa human 222 aa 243 HC aa human 452 aa 244 LC DNA human 666
nucleotide (coding strand) 245 HC DNA human 1356 nucleotide (coding
strand) 242 X910/12-HC-L0-IFN.alpha.(R144A) IgG4 LC aa human 222 aa
246 HC aa synthetic 614 aa 244 LC DNA human 666 nucleotide (coding
strand) 247 HC DNA synthetic 1842 nucleotide (coding strand) 242
X910/12-HC-L0-IFN.alpha.(A145D) IgG4 LC aa human 222 aa 248 HC aa
synthetic 614 aa 244 LC DNA human 666 nucleotide (coding strand)
249 HC DNA synthetic 1842 nucleotide (coding strand) 250 X913/15
IgG1 LC aa human 222 aa 251 HC aa human 450 aa 252 LC DNA human 666
nucleotide (coding strand) 253 HC DNA human 1350 nucleotide (coding
strand) 250 X913/15-HC-L0-IFN.alpha.(R144A) IgG4 LC aa human 222 aa
254 HC aa synthetic 612 aa 252 LC DNA human 666 nucleotide (coding
strand) 255 HC DNA synthetic 1836 nucleotide (coding strand) 250
X913/15-HC-L0-IFN.alpha.(A145D) IgG4 LC aa human 222 aa 256 HC aa
synthetic 612 aa 252 LC DNA human 666 nucleotide (coding strand)
257 HC DNA synthetic 1836 nucleotide (coding strand) 258 R5D1 IgG1
LC aa synthetic 214 aa 259 HC aa synthetic 450 aa 260 LC DNA
synthetic 642 nucleotide (coding strand) 261 HC DNA synthetic 1350
nucleotide (coding strand) 258 R5D1-HC-L0-IFN.alpha.(A145D) IgG4 LC
aa synthetic 214 aa 262 HC aa synthetic 612 aa 260 LC DNA synthetic
642 nucleotide (coding strand) 263 HC DNA synthetic 1836 nucleotide
(coding strand) 264 R5E8 IgG1 LC aa synthetic 219 aa 265 HC aa
synthetic 453 aa 266 LC DNA synthetic 657 nucleotide (coding
strand) 267 HC DNA synthetic 1359 nucleotide (coding strand) 264
R5E8-HC-L0-IFN.alpha.(A145D) IgG4 LC aa synthetic 219 aa 268 HC aa
synthetic 615 aa 266 LC DNA synthetic 657 nucleotide (coding
strand) 269 HC DNA synthetic 1845 nucleotide (coding strand) 270
R10A2 IgG1 LC aa synthetic 214 aa 271 HC aa synthetic 450 aa 272 LC
DNA synthetic 642 nucleotide (coding strand) 273 HC DNA synthetic
1350 nucleotide (coding strand) 270 R10A2-HC-L0-IFN.alpha.(A145D)
IgG4 LC aa synthetic 214 aa 274 HC aa synthetic 612 aa 272 LC DNA
synthetic 642 nucleotide (coding strand) 275 HC DNA synthetic 1836
nucleotide (coding strand) 276 Rituximab LC aa synthetic 213 aa 277
HC aa synthetic 451 aa 278 LC DNA synthetic 639 nucleotide (coding
strand) 279 HC DNA synthetic 1353 nucleotide (coding strand) 276
Rituximab-HC-L6-IFN.alpha. IgG1 LC aa synthetic 213 aa 280 HC aa
synthetic 622 aa 278 LC DNA synthetic 639 nucleotide (coding
strand) 281 HC DNA synthetic 1866 nucleotide (coding strand) 276
Rituximab-HC-L6-IFN.alpha.(R144A) LC aa synthetic 213 aa 282 IgG1
HC aa synthetic 622 aa 278 LC DNA synthetic 639 nucleotide (coding
strand) 283 HC DNA synthetic 1866 nucleotide (coding strand) 276
Rituximab-HC-L6-IFN.alpha.(A145G) LC aa synthetic 213 aa 284 IgG1
HC aa synthetic 622 aa 278 LC DNA synthetic 639 nucleotide (coding
strand) 285 HC DNA synthetic 1866 nucleotide (coding strand) 276
Rituximab-HC-L6-IFN.alpha.(R33A + YNS) LC aa synthetic 213 aa 286
IgG1 HC aa synthetic 622 aa 278 LC DNA synthetic 639 nucleotide
(coding strand) 287 HC DNA synthetic 1866 nucleotide (coding
strand) 276 Rituximab-HC-L6- LC aa synthetic 213 aa 288
IFN.alpha.(R144A + YNS) IgG1 HC aa synthetic 622 aa 278 LC DNA
synthetic 639 nucleotide (coding strand) 289 HC DNA synthetic 1866
nucleotide (coding strand) 290 Palivizumab LC aa synthetic 213 aa
291 HC aa synthetic 450 aa 292 LC DNA synthetic 639 nucleotide
(coding strand) 293 HC DNA synthetic 1350 nucleotide (coding
strand) 290 Palivizumab-HC-L6-IFN.alpha. IgG1 LC aa synthetic 213
aa 294 HC aa synthetic 621 aa 292 LC DNA synthetic 639 nucleotide
(coding strand) 295 HC DNA synthetic 1863 nucleotide (coding
strand) 290 Palivizumab-HC-L6-IFN.alpha. Fab LC aa synthetic 213 aa
296 HC aa synthetic 394 aa 292 LC DNA synthetic 639 nucleotide
(coding strand) 297 HC DNA synthetic 1182 nucleotide (coding
strand) 290 Palivizumab-HC-L6-IFN.alpha.(A145D) LC aa synthetic 213
aa 298 Fab HC aa synthetic 394 aa 292 LC DNA synthetic 639
nucleotide (coding strand) 299 HC DNA synthetic 1182 nucleotide
(coding strand) 300 J110 IgG1 LC aa synthetic 214 aa 301 HC aa
synthetic 449 aa 302 LC DNA synthetic 642 nucleotide (coding
strand) 303 HC DNA synthetic 1347 nucleotide (coding strand) 300
J110-HC-L6-IL-4 IgG1 LC aa synthetic 214 aa 304 HC aa synthetic 584
aa 302 LC DNA synthetic 642 nucleotide (coding strand) 305 HC DNA
synthetic 1752 nucleotide (coding strand) 300 J110-HC-L6-IL-4(R88Q)
IgG1 LC aa synthetic 214 aa 306 HC aa synthetic 584 aa 302 LC DNA
synthetic 642 nucleotide (coding strand) 307 HC DNA synthetic 1752
nucleotide (coding strand) 300 J110-HC-L16-IL-6 IgG1 LC aa
synthetic 214 aa 308 HC aa synthetic 649 aa 302 LC DNA synthetic
642 nucleotide (coding strand) 309 HC DNA synthetic 1947 nucleotide
(coding strand) 300 J110-HC-L16-IL-6(R179E) IgG1 LC aa synthetic
214 aa 310 HC aa synthetic 649 aa 302 LC DNA synthetic 642
nucleotide (coding strand) 311 HC DNA synthetic 1947 nucleotide
(coding strand) 312 HB95 IgG1 LC aa synthetic 215 aa 313 HC aa
synthetic 450 aa 314 LC DNA synthetic 645 nucleotide (coding
strand) 315 HC DNA synthetic 1350 nucleotide (coding strand) 312
HB95-HC-L0-IFN.alpha.(A145D) IgG4 LC aa synthetic 215 aa 316 HC aa
synthetic 612 aa 314 LC DNA synthetic 645 nucleotide (coding
strand) 317 HC DNA synthetic 1836 nucleotide (coding strand) 312
HB95-HC-L6-IFN.alpha. Fab LC aa synthetic 215 aa 318 HC aa
synthetic 394 aa 314 LC DNA synthetic 645 nucleotide (coding
strand) 319 HC DNA synthetic 1182 nucleotide (coding strand) 312
HB95-HC-L6-IFN.alpha.(A145D) Fab LC aa synthetic 215 aa 320 HC aa
synthetic 394 aa 314 LC DNA synthetic 645 nucleotide (coding
strand) 321 HC DNA synthetic 1182 nucleotide (coding strand) 312
HB95-HC-L16-IL-6 IgG1 LC aa synthetic 215 aa 322 HC aa synthetic
650 aa 314 LC DNA synthetic 645 nucleotide (coding strand) 323 HC
DNA synthetic 1950 nucleotide (coding strand) 312
HB95-HC-L16-IL-6(R179E) IgG1 LC aa synthetic 215 aa 324 HC aa
synthetic 650 aa 314 LC DNA synthetic 645 nucleotide (coding
strand) 325 HC DNA synthetic 1950 nucleotide (coding strand) 326
nBT062 IgG1 LC aa synthetic 214 aa 327 HC aa synthetic 452 aa 328
LC DNA synthetic 642 nucleotide (coding
strand) 329 HC DNA synthetic 1356 nucleotide (coding strand) 326
nBT062-HC-L0-IFN.alpha.(A145D) IgG4 LC aa synthetic 214 aa 330 HC
aa synthetic 614 aa 328 LC DNA synthetic 642 nucleotide (coding
strand) 331 HC DNA synthetic 1842 nucleotide (coding strand) 332
C21 IgG1 LC aa synthetic 214 aa 333 HC aa synthetic 448 aa 334 LC
DNA synthetic 642 nucleotide (coding strand) 335 HC DNA synthetic
1344 nucleotide (coding strand) 332 C21-HC-L0-IFN.alpha.(A145D)
IgG4 LC aa synthetic 214 aa 336 HC aa synthetic 610 aa 334 LC DNA
synthetic 642 nucleotide (coding strand) 337 HC DNA synthetic 1830
nucleotide (coding strand) 338 7.1 IgG1 LC aa synthetic 214 aa 339
HC aa synthetic 449 aa 340 LC DNA synthetic 642 nucleotide (coding
strand) 341 HC DNA synthetic 1347 nucleotide (coding strand) 338
7.1-HC-L0-IFN.alpha.(A145D) IgG4 LC aa synthetic 214 aa 342 HC aa
synthetic 611 aa 340 LC DNA synthetic 642 nucleotide (coding
strand) 343 HC DNA synthetic 1833 nucleotide (coding strand) 344
2D12 IgG1 LC aa synthetic 213 aa 345 HC aa synthetic 452 aa 346 LC
DNA synthetic 639 nucleotide (coding strand) 347 HC DNA synthetic
1356 nucleotide (coding strand) 344 2D12-HC-L6-IFN.alpha.(A145G)
IgG1 LC aa synthetic 213 aa 348 HC aa synthetic 623 aa 346 LC DNA
synthetic 639 nucleotide (coding strand) 349 HC DNA synthetic 1869
nucleotide (coding strand) 344 2D12-HC-L6-IFN.alpha.(A145G) IgG4 LC
aa synthetic 213 aa 350 HC aa synthetic 620 aa 346 LC DNA synthetic
639 nucleotide (coding strand) 351 HC DNA synthetic 1860 nucleotide
(coding strand) 344 2D12-HC-L0-IFN.alpha.(A145D) IgG4 LC aa
synthetic 213 aa 352 HC aa synthetic 614 aa 346 LC DNA synthetic
639 nucleotide (coding strand) 353 HC DNA synthetic 1842 nucleotide
(coding strand) 344 2D12-HC-L6-IFN.alpha. Fab LC aa synthetic 213
aa 354 HC aa synthetic 396 aa 346 LC DNA synthetic 639 nucleotide
(coding strand) 355 HC DNA synthetic 1188 nucleotide (coding
strand) 344 2D12-HC-L6-IFN.alpha.(A145D) Fab LC aa synthetic 213 aa
356 HC aa synthetic 396 aa 346 LC DNA synthetic 639 nucleotide
(coding strand) 357 HC DNA synthetic 1188 nucleotide (coding
strand) 344 2D12-HC-L6-IL-4(R88Q) IgG1 LC aa synthetic 213 aa 358
HC aa synthetic 587 aa 346 LC DNA synthetic 639 nucleotide (coding
strand) 359 HC DNA synthetic 1761 nucleotide (coding strand) 344
2D12-HC-L16-IL-6 IgG1 LC aa synthetic 213 aa 360 HC aa synthetic
652 aa 346 LC DNA synthetic 639 nucleotide (coding strand) 361 HC
DNA synthetic 1956 nucleotide (coding strand) 344
2D12-HC-L16-IL-6(R179E) IgG1 LC aa synthetic 213 aa 362 HC aa
synthetic 652 aa 346 LC DNA synthetic 639 nucleotide (coding
strand) 363 HC DNA synthetic 1956 nucleotide (coding strand) 364
X355/01 IgG1 LC aa human 214 aa 365 HC aa human 455 aa 366 LC DNA
human 642 nucleotide (coding strand) 367 HC DNA human 1365
nucleotide (coding strand) 368 X355/04 IgG1 LC aa human 219 aa 369
HC aa human 453 aa 370 LC DNA human 657 nucleotide (coding strand)
371 HC DNA human 1359 nucleotide (coding strand) 372 R10B10 IgG1 LC
aa synthetic 220 aa 373 HC aa synthetic 449 aa 374 R7H11 IgG1 LC aa
synthetic 220 aa 375 HC aa synthetic 449 aa 376 R7F11 IgG1 LC aa
synthetic 214 aa 377 HC aa synthetic 452 aa 276
Rituximab-HC-L7-IFN.gamma.(.DELTA.[A23,D24]) LC aa synthetic 213 aa
378 IgG1 HC aa synthetic 599 aa 278 LC DNA synthetic 639 nucleotide
(coding strand) 379 HC DNA synthetic 1797 nucleotide (coding
strand) 226 X355/02-HC-L7-IFN-.gamma.(S20I) IgG1 LC aa human 222 aa
380 HC aa synthetic 601 aa 228 LC DNA human 666 nucleotide (coding
strand) 381 HC DNA synthetic 1803 nucleotide (coding strand) 270
R10A2-HC-L7-IFN.gamma.(D21K) IgG1 LC aa synthetic 214 aa 382 HC aa
synthetic 600 aa 272 LC DNA synthetic 642 nucleotide (coding
strand) 383 HC DNA synthetic 1800 nucleotide (coding strand) 276
Rituximab-HC-L6-IFN.alpha.(R33A) IgG1 LC aa synthetic 213 aa 436 HC
aa synthetic 622 aa 278 LC DNA synthetic 639 nucleotide (coding
strand) 437 HC DNA synthetic 1866 nucleotide (coding strand)
TABLE-US-00042 TABLE 39 Variable Domains SEQ ID Length NO: Clone
Antigen Chain Species (aa) 384 G005 CD38 V.kappa. human 107 385
G005 CD38 VH human 122 386 MORAB03080 CD38 V.lamda. human 106 387
MORAB03080 CD38 VH human 122 388 hu38SB19 CD38 V.kappa. synthetic
107 (SAR650984) 389 hu38SB19 CD38 VH synthetic 120 (SAR650984) 390
X355/02 CD38 V.lamda. human 116 391 X355/02 CD38 VH human 121 392
X355/07 CD38 V.kappa. human 108 393 X355/07 CD38 VH human 118 394
X910/12 CD38 V.lamda. human 116 395 X910/12 CD38 VH human 122 396
X913/15 CD38 V.lamda. human 116 397 X913/15 CD38 VH human 120 398
R5D1 CD38 V.kappa. rat 107 399 R5D1 CD38 VH rat 120 400 R5E8 CD38
V.kappa. rat 112 401 R5E8 CD38 VH rat 123 402 R10A2 CD38 V.kappa.
rat 107 403 R10A2 CD38 VH rat 120 404 Rituximab CD20 V.kappa. mouse
106 405 Rituximab CD20 VH mouse 121 406 Palivizumab Respiratory
V.kappa. synthetic 106 Syncytial Virus (RSV) 407 Palivizumab RSV VH
synthetic 120 408 J110 PD-1 V.kappa. mouse 107 409 J110 PD-1 VH
mouse 119 410 HB95 HLA V.kappa. mouse 108 411 HB95 HLA VH mouse 120
412 nBT062 CD138 V.kappa. mouse 107 413 nBT062 CD138 VH mouse 122
414 C21 High Molecular V.lamda. synthetic 108 Weight Melanoma-
Associated Antigen (HMW-MAA) 415 C21 HMW-MAA VH synthetic 118 416
7.1 HMW-MAA V.kappa. mouse 107 417 7.1 HMW-MAA VH mouse 119 418
2D12 Yellow Fever V.kappa. mouse 106 Virus (YFV) 419 2D12 YFV VH
mouse 122 420 X355/01 CD38 V.kappa. human 107 421 X355/01 CD38 VH
human 125 422 X355/04 CD38 V.kappa. human 112 423 X355/04 CD38 VH
human 123 424 R10B10 CD38 V.lamda. rat 114 425 R10B10 CD38 VH rat
119 426 R7H11 CD38 V.lamda. rat 114 427 R7H11 CD38 VH rat 119 428
R7F11 CD38 V.kappa. rat 107 429 R7F11 CD38 VH rat 122
TABLE-US-00043 TABLE 40 Other Single Polypeptide Chain Sequences
SEQ ID NO: Species Length Gene 430 human 297 CD20 431 human 288
PD-1 432 human 310 CD138 433 human 2322 High Molecular Weight
Melanoma-Associated Antigen (HMW-MAA) 434 human 165 IFN.alpha.2c
435 human 166 IFN.alpha.4a
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160367695A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160367695A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References