NZ623812B2 - Polypeptide constructs and uses thereof - Google Patents
Polypeptide constructs and uses thereof Download PDFInfo
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- NZ623812B2 NZ623812B2 NZ623812A NZ62381212A NZ623812B2 NZ 623812 B2 NZ623812 B2 NZ 623812B2 NZ 623812 A NZ623812 A NZ 623812A NZ 62381212 A NZ62381212 A NZ 62381212A NZ 623812 B2 NZ623812 B2 NZ 623812B2
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Abstract
Disclosed is a polypeptide construct comprising a peptide or polypeptide signalling 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 and wherein the peptide or polypeptide signalling ligand is selected from the group consisting of an IFN, IL-4 and IL-6. y on cells lacking expression of said antigen and wherein the peptide or polypeptide signalling ligand is selected from the group consisting of an IFN, IL-4 and IL-6.
Description
POLYPEPTIDE CONSTRUCTS AND USES THEREOF
RELATED APPLICATIONS
This application claims benefit of Australian patent application 2011904502
entitled “Polypeptide constructs and uses thereof”, filed 28 October 2011, the entire
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
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
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.
[0004] 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)-a 2b, IFNa 2a, IFNb , 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 IFNa 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
9293452-1
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.
Even though some ligands, such as IFNa 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.
Similarly, it has been known since the mid-1980’s that interferons, in particular
IFNa , 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. IFNa
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 IFNa on the tumour cells is mediated
by the IFNa binding directly to the type I IFN receptor on those cells and stimulating
apoptosis, terminal differentiation or reduced proliferation. One “indirect” effect of IFNa
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.
Unfortunately, the type I interferon receptor is also present on most non-
cancerous cells. Activation of this receptor on such cells by IFN a causes the expression of
numerous pro-inflammatory cytokines and chemokines, leading to toxicity. Such toxicity
prevents the dosing of IFNa to a subject at levels that exert the maximum anti-proliferative
and pro-apoptotic activity on the cancer cells.
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Ozzello et al. (Breast Cancer Research and Treatment 25:265-76, 1993)
described covalently attaching human IFNa to a tumour-targeting antibody, thereby
localizing the direct inhibitory activity of IFNa 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 IFNa on the cancer cells, since the human IFNa 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 IFNa to the murine cells, however, the authors could not evaluate the toxicity of
the antibody-IFNa conjugate relative to free INFa . These authors used a chemical method
to attach the IFNa to the antibody.
Alkan et al., (Journal of Interferon Research, volume 4, number 3, p. 355-63,
1984) demonstrated that attaching human IFNa 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 IFNa to an antibody could be used as a treatment for
cancer since it would reduce tumour growth. Alkan et al did not address the potential
toxicity of these antibody-IFNa conjugates arising from their interactions with normal,
antigen-negative cells.
It is also known that the linkage between an antibody and IFNa may be
accomplished by making a fusion protein construct. For example, IDEC (WO01/97844)
disclose a direct fusion of human IFNa 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 IFNa . For example, US 7,456,257
discloses that the C-terminus of an antibody heavy chain constant region may be connected
to IFNa via an intervening serine-glycine rich (S/G) linker of the sequence (GGGGS) ,
where n may be 1, 2 or 3, and that there are no significant differences in the IFN a activity
of the fusion protein construct regardless of linker length.
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Morrison et al. (US2011/0104112 A1; and Xuan C, Steward KK, Timmerman
JM, Morrison SL. Targeted delivery of interferon-α via fusion to anti-CD20 results in
potent antitumor activity against B-cell lymphoma. Blood 2010;115:2864–71) also
disclose IFNa 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 IFNa reduced the activity of IFNa 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 IFNa (free IFNa ) acting on
human cells, but appeared to be more significant for murine IFNa on murine cells. The
decrease in the activity of human IFNa that results from fusing it to the C-terminus of an
antibody, as observed by Morrison et al, and in US 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 al (Blood vol. 114, No. 18, pp3864-71), who
used an alternative strategy of attaching the IFNa to a tumor targeting antibody in such a
way that no loss in IFNa activity was observed.
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 IFNa portion of the antibody-IFNa fusion protein
maintained a high proportion of native IFNa 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
IFNa 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 IFNa 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 IFNa to the cancer cells in such a way that it has dramatically more activity
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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
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.
[0014] 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.
[0015] 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.
In a third aspect, the present invention provides use of the polypeptide construct
of the present invention in the treatment of cancer.
[0017] 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.
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
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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.
Unlike the linking of a non-attenuated “native” or “wild-type” human ligand to an
antibody or antigen-binding portion therof, 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.
In one embodiment the signaling ligand is IFNα or IFNβ 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.
The present invention also provides an antibody-attenuated ligand fusion proteins,
wherein the attenuated ligand is IFNa or IFNb and the wherein fusion protein construct,
when injected into a mouse with an established human tumor, can eliminate the tumor.
The present invention also provides an antibody-attenuated ligand fusion proteins,
wherein the attenuated ligand is IFNa or IFNb 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.
The present invention also provides an antibody-attenuated ligand fusion proteins,
wherein the attenuated ligand is IFNa or IFNb 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.
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An antibody-attenuated ligand fusion proteins, wherein the attenuated ligand is
IFNa or IFNb and wherein the fusion protein construct can eliminate both established
myeloma tumors and established lymphoma tumors in a mouse
In each of these cases it is preferred that cell surface-associated antigen is CD 38.
[0026] 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.
In one embodiment, the construct is a fusion protein.
[0028] 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).
In one embodiment the cell surface associated antigen is selected from class I
MHC or PD-1.
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 IFNα.
In one embodiment, the signaling ligand is selected from any one of IFNα2b,
IFNβ, IL-4 or IL-6.
[0032] In certain embodiments in which the signaling ligand is an IFNα, 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 IFNα 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
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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).
In certain embodiments in which the signaling ligand is an IFNα and the cell
surface associated antigen is CD38, the antibody is selected from any one of G003, G005,
G024, MOR03077, MORO3079, MORO3080, MORO3100, 38SB13, 38SB18, 38SB19,
38SB30, 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.
In certain embodiments in which the cell surface associated antigen is CD38, the
signaling ligand of the polypeptide construct is an IFNα, 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.
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.
[0036] In certain embodiments in which the signaling ligand of the polypeptide construct
is an IFNα, the antibody or antigen binding portion thereof may bind to a cell surface
asociated 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.
In certain embodiments in which the signaling ligand of the polypeptide construct
is IFNa or IFNβ, the cell surface associated antigen is selected from CD20, CD38, CD138
or CS1. In certain embodiments in which the ligand is IFNa or IFNβ, the tumour or cancer
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may be selected from multiple myeloma, melanoma, renal cell carcinoma, chronic
myelogenous leukemia or hairy cell leukemia.
In a particular embodiment the construct is G005-HC-L0-IFNα (A145D) IgG4.
In certain embodiments in which the signaling ligand of the polypeptide constrct
is an IFNβ, the cell surface associated antigen may be a T cell, a myeloid cell or an antigen
presenting cell cell surface associated protein.
In certain embodiments in which the signaling ligand of the polypeptide construct
is an IFNβ, 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 embodimens, the
construct may be used to treat a disease characterized by excess inflamammation, such as
an autoimmune disease.
In certain embodiments in which the signaling ligand of the polypeptide construct
is an IFNβ, 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 IFNβ may also possess a C17S or C17A substitution.
In certain embodiments the signaling ligand of the polypeptide construct is an
IFNγ. 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.
In certain embodiments in which the signaling ligand of the polypeptide construct
is an IFNγ, 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.
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.
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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.
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.
In certain embodiments in which the signaling ligand of the polypeptide construct
is a TGFβ, 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.
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.
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).
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:
Name V sequence V /V sequence
H K L
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
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Name V sequence V /V sequence
H K L
R5E8 SEQ ID NO:401 SEQ ID NO:400
R10A2 SEQ ID NO:403 SEQ ID NO:402
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
Figure 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.
[0053] Figure 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.
[0054] Figure 3 shows the amino acid sequences of the human CD38 (SEQ ID NO:131).
Figure 4 shows the amino acid sequences of certain exemplary signaling ligands
of the present invention: (a) human IFNa 2b, IFNb 1, IFNb 1b and IFNg ; (b) IL-4 and IL-6.
Figure 5 shows the amino acid sequences of certain antibody-attenuated ligand
fusion proteins of the present invention: (a) G005-HC-L0-IFNα (A145D) IgG4; (b)
nBT062-HC-L0-IFNα (A145D) IgG4; (c) G005-HC-L0-IFNβ (R35A) IgG4; (d) HB95-
HC-L16-IL-6 (R179E) IgG1; and (e) J110-HC-L6-IL-4 (R88Q) IgG1. The nomenclature
for the fusion proteins is described in the examples.
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Figure 6 shows the non-antibody-antigen-targeted interferon activity of IFNa 2b,
and of the antibody-IFN fusion protein constructs Rituximab-IFNa 2b (Rituximab-HC-L6-
IFNa IgG1) and Palivizumab-IFNa 2b (Isotype-HC-L6-IFNa IgG1) in the interferon
activity assay described in the examples below as the “off-target assay. Throughout the
figures “IFNa 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.
Figure 7 shows the antibody-antigen-targeted interferon activity of the
Rituximab-IFNa 2b fusion protein construct (Rituximab-HC-L6-IFNa IgG1) compared
with IFNa 2b in the antiproliferative assay described in the examples below as the “on
target (Daudi) assay.”
Figure 8 shows the antibody-antigen-targeted interferon activity of the
Rituximab-IFNa fusion protein construct (Rituximab-HC-L6-IFNa IgG1) compared with
the non-targeted activity of Palivizumab-IFNa fusion protein construct (Isotype-HC-L6-
IFNa IgG1) in the “on-target (Daudi) assay” described in the examples below.
Figure 9 shows the non-antibody-antigen-targeted interferon activity of IFNa 2b,
of the antibody-IFN fusion protein constructs Rituximab-IFNa 2b (Rituximab-HC-L6-IFNa
IgG1) and Palivizumab-IFNa 2b (Isotype-HC-L6-IFNa IgG1), and of certain variants of
Rituximab-IFNa 2b constructs that have been mutated to reduce their interferon activity.
The assay is described in the examples as the “off-target assay”.
Figure 10 shows the non-antibody-antigen-targeted interferon activity of the
antibody-IFN fusion protein constructs Rituximab-IFNa 2b (Rituximab-HC-L6-IFNa
IgG1) and of two variants of Rituximab-IFNa 2b that were mutated to reduce interferon
activity. The assay is described in the examples as the “off-target assay”.
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Figure 11 shows the antibody-antigen-targeted interferon activity of the antibody-
IFN fusion protein construct Rituximab-IFNa 2b (Rituximab-HC-L6-IFNa IgG1) and of
variants of Rituximab-IFNa 2b constructs that have been mutated to reduce their interferon
activity compared to the non-targeted activity of the Palivizumab-IFNa 2b (Isotype-HC-
L6-IFNa IgG1) fusion protein constructs and compared to IFNa 2b. The assay is described
in the examples as the “on target (Daudi) assay.”
Figure 12 shows the antibody-antigen-targeted interferon activity of the antibody-
IFN fusion protein constructs Rituximab-IFNa 2b (Rituximab-HC-L6-IFNa 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.”
Figure 13 shows the sequences of certain novel human CD38 antibodies
disclosed herein.
Figure 14 shows the results of detection of binding of novel human anti-CD38
antibodies to a CD38 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.
Figure 15 shows the non-antibody-antigen targeted IFN activity of IFNa 2b
compared with an anti-CD38-IFNa fusion protein construct (G005-HC-L0-IFNa IgG4),
based on the anti-CD38 antibody G005. The assay is described in the examples as the “off-
target assay.”
[0067] Figure 16 shows the antiproliferative activity of IFNa 2b vs an anti-CD38-IFNa
fusion protein construct (G005-HC-L0-IFNa IgG4) on the multiple myeloma cell line
ARP-1 (CD38 ). The assay is described in the examples as the “on target (ARP) assay.”
Figure 17 shows the non-antibody-antigen targeted IFN activity of IFNa 2b vs
various anti-CD38-IFNa fusion protein constructs bearing point mutations in the IFN
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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.”
Figure 18 shows the non-antibody-antigen targeted IFN activity of IFNa 2b vs
various anti-CD38-IFNa 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.”
Figure 19 shows the non-antibody-antigen targeted IFN activity of IFNa 2b vs
two anti-CD38-IFNa 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.”
Figure 20 shows the antiproliferative activity of IFNa 2b vs anti-CD38-IFNa
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.”
[0072] Figure 21 shows the anti-proliferative activity of IFNa 2b and various anti-CD38-
IFNα 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”.
[0073] Figure 22 shows the anti-proliferative activity of IFNa 2b and two anti-CD38-
IFNa 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.”
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Figure 23 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa 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.”
Figure 24 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa 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.”
[0076] Figure 25 shows the non-antibody-antigen targeted IFN activity of various anti-
CD38-IFNa 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.”
Figure 26 shows the non-antibody-antigen targeted IFN activity of various anti-
CD38-IFNa 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.”
Figure 27 shows the non-antibody-antigen targeted IFN activity of various anti-
CD38-IFNa 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.”
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Figure 28 shows the non-antibody-antigen targeted IFN activity of various anti-
CD38-IFNa 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] Figure 29 shows the non-antibody-antigen targeted IFN activity of IFNa 2b vs
various anti-CD38-IFNa 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.”
Figure 30 shows the non-antibody-antigen targeted IFN activity of various anti-
CD38-IFNa 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.”
Figure 31 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of anti-CD38-IFNa 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.”
Figure 32 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa 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.”.
Figure 33 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa 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.”
Figure 34 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa fusion protein constructs with the A145D mutation
in the IFN portion. The experiment compares different antibody variable regions in the
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context of the same mutated IFN fusion protein construct. The assay is described in the
examples as the “on target (ARP) assay.”
Figure 35 shows the non-antibody-antigen targeted IFN activity of IFNa 2b and
various anti-CD38-IFNa 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.”
Figure 36 shows the non-antibody-antigen targeted IFN activity of IFNa 2b and
various anti-CD38-IFNa 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.”
Figure 37 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNa 2b vs anti-CD38-IFNa 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.”
Figure 38 shows the non-antibody-antigen targeted IFN activity of IFNa 2b and
various anti-CD38-IFNa 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.”
Figure 39 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of two antibody-IFNa 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.”
Figure 40 (a) shows the antiproliferative activity on the multiple myeloma cell
line ARP-1 of IFNa 2b and two antibody-IFNa fusion protein constructs with the A145D
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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. (b) shows
the same assay, comparing antibody-attenuated IFNα fusion protein constructs in which
the antibody portion is a Fab fragment rather than a full size antibody. For both (a) and (b)
panels, the assay is described in the examples as the “on target (ARP) assay.”
Figure 41 shows measurements of the antiviral activity of IFNa and two
antibody-IFNa 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.
Figure 42 shows the non-antibody-antigen targeted IFN activity of IFNb , an anti-
CD38-IFNb 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.”
Figure 43 shows the antiproliferative activity on the multiple myeloma cell line
ARP-1 of IFNb , an anti-CD38-IFNb 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.
Figure 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.
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Figure 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.
Figure 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.
[0098] Figure 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.
Figure 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.
Figure 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.
Figure 50 shows the effects of an anti-CD38-attenuated IFNa fusion protein
construct (G005-HC-L6-IFNa (A145G) IgG1) and an isotype control-attenuated IFNa
fusion protein construct (Isotype-HC-L6-IFNa (A145G) IgG1) on the growth of
subcutaneous H929 myeloma tumors in SCID mice, at various doses. The bar labeled
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“treatment” shows the duration of treatment with the compounds. The “isotype” antibody
was based on antibody 2D12.
Figure 51 shows the effects of anti-CD38-attenuated IFNa fusion protein
constructs vs isotype control antibody-attenuated IFNa 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.
Figure 52 shows the effects of an anti-CD38-attenuated IFNa fusion protein
construct (X355/02-HC-L0-IFNa (A145D) IgG4) vs an isotype control antibody-
attenuated IFNa 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.
Figure 53 shows the effects of various compounds on the growth of subcutaneous
H929 myeloma tumors in SCID mice. G005 is an anti-CD38 antibody.
Figure 54 shows the effects of an anti-CD38-attenuated IFNa fusion protein
construct (G005-HC-L6-IFNa (A145G) IgG4) and an isotype control-attenuated IFNa
fusion protein construct (Isotype-HC-L6-IFNa (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.
Figure 55 shows the effects of an anti-CD38-attenuated IFNa fusion protein
construct (G005-HC-L6-IFNa (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 .
[0107] Figure 56 shows the inhibition of colony formation from normal human bone
marrow mononuclear cells (BM MNC) by IFNa2b, an anti-CD38-attenuated IFNa fusion
protein construct (G005-HC-L0-IFNa (A145D) IgG4) and an isotype control antibody-
attenuated IFNa fusion protein construct 2D12-HC-L0-IFNa (A145D) IgG4. The
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antibody-attenuated IFNa fusion protein constructs show about 10,000-fold reduced
potency in this assay.
Figure 57 shows the effects of IFNa 2b vs an antibody-attenuated IFNa fusion
protein construct (Isotype-HC-L6-IFNa (A145G) IgG1; the isotype variable regions are
based on antibody 2D12) on cytokine production by human peripheral blood mononuclear
cells (PBMCs). (a) IP-10 and MCP-1; (b) MCP-3 and IL-1a .
DETAILED DESCRIPTION OF THE INVENTION
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).
“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
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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 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 examplefied 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 naïve CD4+ T cells into Th1, Th17, Th2 vs.
Treg cells), increases or decreases in gene expression or gene product secretion into the
media, etc.
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.
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.
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.
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
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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.
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.
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: FRl, CDRl, 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, IgAl and IgA2) or subclass.
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)
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(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 U S A 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
41354-5). In an embodiment the antibody binding portion is a Fab fragment.
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.,
US5225539, US6054297, US7566771 or US5585089. The term “humanized antibody”
also encompasses a super-humanized antibody, e.g., as described in US7,732,578.
9293452-1
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 US5,565,332). 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
US6300064 and/or US6248516.
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.
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).
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).
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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. Patent No 5,837,821.
[0124] 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.
[0125] 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; US 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 α-crystallin or ubiquitin molecule (Affilin); trypsin inhibitor II (Microbody); α2p8
or Ankyrin repeat (repeat-motif proteins), Charybdotoxin (Scorpion toxins), Min-23,
Cellulose binding domain (Knottins); Neocarzinostatin, CBM4-2 and Tendamistat.
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.
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,
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polypeptide or protein synthesis and the use of crosslinkers and other methods which
impose conformational constraints on the proteinaceous molecule or their analogues.
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 ; 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 pyridoxalphosphate followed by reduction with NaBH .
[0129] 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.
The carboxyl group may be modified by carbodiimide activation via O-
acylisourea formation followed by subsequent derivatisation, for example, to a
corresponding amide.
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-
chloromercurinitrophenol and other mercurials; carbamoylation with cyanate at alkaline
Tryptophan residues may be modified by, for example, oxidation with N-
bromosuccinimide or alkylation of the indole ring with 2-hydroxynitrobenzyl bromide
or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by
alkylation with iodoacetic acid derivatives or N-carbethoxylation with
diethylpyrocarbonate.
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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-hydroxyphenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline,
phenylglycine, ornithine, sarcosine, 4-aminohydroxymethylheptanoic 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 1
________________________________________________________________________
Non-conventional Code Non-conventional Code
amino acid amino acid
________________________________________________________________________
α-aminobutyric acid Abu L-N-methylalanine Nmala
α-amino-α-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 α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl-γ-aminobutyrate Mgabu
40 D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa
D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen
D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap
D-α-methylaspartate Dmasp α-methylpenicillamine Mpen
D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
45 D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
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D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu
D-α-methylleucine Dmleu α-napthylalanine Anap
D-α-methyllysine Dmlys N-benzylglycine Nphe
D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-α-methylserine Dmser N-cyclobutylglycine Ncbut
D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-α-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-γ-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
γ-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-α-methylalanine Mala
L-α-methylarginine Marg L-α-methylasparagine Masn
L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug
40 L-α-methylcysteine Mcys L-methylethylglycine Metg
L-α-methylglutamine Mgln L-α-methylglutamate Mglu
L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe
L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-α-methylleucine Mleu L-α-methyllysine Mlys
45 L-α-methylmethionine Mmet L-α-methylnorleucine Mnle
L-α-methylnorvaline Mnva L-α-methylornithine Morn
L-α-methylphenylalanine Mphe L-α-methylproline Mpro
L-α-methylserine Mser L-α-methylthreonine Mthr
L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr
50 L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
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N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe
carbamylmethyl)glycine carbamylmethyl)glycine
1-carboxy(2,2-diphenyl- Nmbc
ethylamino)cyclopropane
_______________________________________________________________________
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).
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.
An example of glyco-engineering used the Potelligent® method as described in
Shinkawa T. et al., 2003 (J Biol Chem 278: 3466-73).
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 Qβ 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
9293452-1
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 US patent 6,180,370 or US patent 5,225,539).
[0139] 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 .
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.
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.
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
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
. 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.
9293452-1
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 .
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.
[0145] 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.
[0146] 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.
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,
9293452-1
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.
[0148] 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)
9293452-1
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).
[0149] 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.
[0150] CD38 is a 46kDa 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.)
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 and NADP into cADPR, ADPR and NAADP, depending on
extracellular pH. These products induce Ca -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
9293452-1
including Ca mobilization, cell activation, proliferation, differentiation and migration
(reviewed in Deaglio, S., Trends in Mol. Med. 14:210, 2008.)
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 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.
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)
x y xy
and Lewis , lewis and lewis 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 [US 7,202,346],3A5 [US 7,723,485]).NMB (US
9293452-1
8,039,593), malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science
245:301-304); high molecular weight melanoma antigen (HMW-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 found in colorectal cancer, TRA85
156-22
(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 )
found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Le ),
G49 found in A431 cells, 19.9 found in colon cancer; gastric cancer mucins; R found in
melanoma, MH2 (blood group ALe /Le ) found in colonic adenocarcinoma, 4.2, D1.1,
OFA-1, G , 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,
9293452-1
Crit Rev Immunol.;24:267,2004). Accordingly it may be a potential target cell surface
associated antigen.
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), MART1 (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 (US 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).
[0155] 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
9293452-1
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 2
CEA Antibodies
Ab Clones Patent Assignee Comments
The Dow Chemical
COL-1 US 6,417,337 Humanized
Company
806.077 US 6,903,203 AstraZeneca UK Ltd. Humanized
T84.66 US 7,776,330 City of Hope Humanized
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).
9293452-1
Antibodies to CD33 include, for example, HuM195 (see, e.g., Kossman et al.
(1999) Clin. Cancer Res. 5: 2748-2755; US5693761) and CMA-676 (see, e.g., Sievers et
al., (1999) Blood 93: 3678-3684).
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).
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.
Illustrative anti-HM1.24 antibodies include, but are not limited to a mouse
monoclonal anti-HM1.24 and a humanized anti-HM1.24 IgG1kappa antibody (see, e.g.,
Ono et al. (1999) Mol. Immuno. 36: 387-395).
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™; 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.
,677,171.
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™.,
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;
,770,195, and 5,677,171.
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,
9293452-1
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.
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
,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).
[0165] 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)
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 240kD.
9293452-1
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.
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.
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).
discloses an antibody designated "SC104" together with a range
of humanised variants which bind an antigen expressed on a range of tumour cells.
[0171] 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,
9293452-1
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.
While the large majority of antibodies tested showed efficient targeting of
attenuated IFNa the present inventors identified examples of two antigens where targeting
attenuated IFNa 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 HMW-MAA, high molecular weight melanoma-associated
antigen). We tested two different anti-HMW-MAA-antibody-IFNa 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. 240kD -450kD 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
240kD.
A second example of an antibody-attenuated IFNa 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 IFNa 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
9293452-1
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.
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 IFNα. 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 ~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.
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 3
Target Examples of Ab in Sequence citation Clinical trial
preclinical or clinical reference
development
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
9293452-1
Target Examples of Ab in Sequence citation Clinical trial
preclinical or clinical reference
development
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
bb 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
9293452-1
Target Examples of Ab in Sequence citation Clinical trial
preclinical or clinical reference
development
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
aa 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;
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:
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Table 4
Company Clone names Sequence citation Ref
Genmab/ G003. G005, G024 W0 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
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
9293452-1
include, but are not limited to cytokines, chemokines, growth factors, hormones,
neurotransmitters, and apoptosis inducing factors.
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, Il-35 and their subfamiles; the interferon (IFN) subfamily
including Interferon type I (IFN-α (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7,
IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21), IFN-β (IFN-β1 (IFNB1)
and IFN-β3 (IFNB3)), IFN-ω ((IFNW1), IFNWP2, IFNWP4, IFNWP5, IFNWP9,
IFNWP15, IFNWP18, and IFNWP19 and IFNK), Interferon type II (IFN-γ) 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-1α, IL-1β, 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 IFNα, IFNα2b, IFNβ1, IFNβ1b
and IFNγ. Preferably the sequence of IFNα is selected from SEQ ID NOs 1 to 3, 80 to 90,
434 and 435.
[0179] Exemplary chemokines include, for example, RANTES, MCAF, MIP1-alpha, IP-
, monocyte chemoattractant protein-1 (MCP-1 or CCL2), interleukin-8 (IL-8), CXCL13,
XCL1 (lymphotactin-α), XCL2 (lymphotactin-β) and fractalkine (CX CL1).
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-α), Transforming growth factor beta(TGF-β),
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Tumor_necrosis_factor-alpha(TNF-α), Vascular endothelial growth factor (VEGF),
placental growth factor (PlGF), IL Cofactor for IL-3 and IL-6, IL T-cell growth
factor, IL-3, IL-4, IL-5, IL-6 and IL-7.
Exemplary apoptosis inducing factors include FasL and TRAIL.
[0182] 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.
[0183] 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),
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.
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.
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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.
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.
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.
In one aspect of the present invention, the ligand is an interferon, examples of
which are type I interferons (IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta),
IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin), type
II interferons (IFN-g ) or type III interferons (IFN-l 1, IFN-l 2 and IFN-l 3) (Pestka,
Immunological Reviews 202(1):8-32, 2004).
Type I interferons all signal through the Type I interferon receptor, which is
made of 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
IFN1 a b, 2 a ,a 2 a b, 4 a b , 5 a , 6 a , 7 a , 8 a , 1 a 0, 1 a a/13, 1 a 4, 1 a 6, 1 a 7, nda 2 a 1, a 2c and
a 4a. Some embodiments comprise IFNa 2b, the sequence of which, SEQ ID NO:3, is
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shown in Figure 4. IFNs have been approved in several forms for several indications, as
outlined in Table 5 (which also shows lists of approved IFNb and g 's):
Table 5
Generic Name Trade name Approved for treatment
Interferon alpha 2a Roferon A Hep C, CML, Hairy cell
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 Multiferon Melanoma, viral and
Interferon malignant disease
(HuIFN-a -Le)
Interferon beta 1a, liquid Rebif Multiple Sclerosis
Interferon beta 1a, Avonex Multiple Sclerosis
lyophylized
Interferon beta 1a, Cinnovex Multiple Sclerosis
biogeneric (Iran)
Interferon beta 1b Betaseron/Betaferon Multiple Sclerosis
Interferon beta 1b, Ziferon Multiple Sclerosis
biosimilar (Iran)
PEGylated interferon Pegasys Hepatitis B and C
alpha 2a
PEGylated interferon Reiferon Retard Hep C, Hep B, Hairy cell ,
alpha 2a (Egypt) melanoma, leukemia, NHL,
Kaposi’s sarcoma
PEGylated interferon PegIntron Hepatitis and melanoma
alpha 2b
PEGylated interferon Pegetron Hepatitis C
alpha 2b plus ribavirin
(Canada)
Interferon alfacon-1 Infergen Hepatitis C
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Generic Name Trade name Approved for treatment
Interferon alpha n3 Alferon N Genital warts
Interferon gamma Actimmune Chronic granulomatous
disease
Non-limiting examples of mutations in IFNa 2b that can be used to reduce its
potency are described in Tables 6 and 7, based on the sequence of human IFNa 2b (SEQ
ID NO:3):
Table 6. Relative biological activities of interferon mutants
relative anti-viral relative anti-
activity proliferative activity
IFNa 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
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relative anti-viral relative anti-
activity proliferative activity
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
∆(L161-E165) 0.72 1.1
Table 7. Relative affinity of interferon mutants to their receptors
Affinity to Affinity to
IFNAR1 IFNAR2
IFNa 2b wild type 1 1
L15A 0.079
A19W 0.82
R22A 0.73
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Affinity to Affinity to
IFNAR1 IFNAR2
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
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Affinity to Affinity to
IFNAR1 IFNAR2
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
∆(161-165) 0.53
These mutants have known reductions in binding to the type 1 interferon receptor
IFNAR1 or IFNAR2, and/or have known reductions in IFNa potency based on cell-based
assays.
The data in these tables was disclosed in the following references:
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.
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-a 2
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mutant with enhanced affinity to IFNAR1 is functionally similar to IFN-b .
Mol. Cell. Biol. 26: 1888-1897.
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.
Kalie, Eyal, Jaitin, Diego A., Abramovich, Renne, Schreiber, Gideon (2007). An
interferon a 2 mutant optimized by phage display for IFNAR1 binding
confers specifically enhanced antitubor activities. J. Biol. Chem. 282:
11602-11611.
Pan, Manjing, Kalie, Eyal, Scaglione, Brian J., Raveche, Elizabeth S., Schreiber,
Gideon, Langer, Jerome A. (2008). Mutation of te IFNAR-1 receptor
binding site of human IFN-a 2 generates Tyep I IFN competitive
antagonists. Biochemistry 47: 12018-12027.
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.
The abbreviation “YNS” is sometimes used herein to represent IFNa variants
including the following mutation: H57Y, E58N and Q61S.
The present invention also contemplates combinations of the abovementioned
mutations or deletions in IFNa .
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
9293452-1
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).
[0197] 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.
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).
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),
9293452-1
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.
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
9293452-1
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-a 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 IFNa 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.
In addition, while the above constructs are based on IFNa 2b, the mutations or
deletions could also be made in the context of any of the other IFNa s or IFNb . In another
embodiment of the present invention, the type I IFN is an IFNb . IFN-β is approved for the
treatment of multiple sclerosis (MS). IFN-b could be attenuated by mutation or deletion
and then attached to an antibody that targets cells involved in the pathogenesis of this
disease. IFN-β 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-β
activity directly to pathogenic cells, these adverse events may be avoided.
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).
9293452-1
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).
In an embodiment of the invention, an attenuated version of IFN-β 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-β may be
effective. Direct effects of IFN-β 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).
Therefore, in one aspect of the present invention, an attenuated IFN-β 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-β
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.
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Table 8
CD3 Antibodies
Ab Clones Patent Assignee Comments
TF NSO
US 7,994,289 BTG International Humanized
CD3A.122
M291 US 7,381,803 PDL BioPharma Humanized
28F1, 27H5,
US 7,728,114 Novimmune S.A. Human
23F10, 15C3
Alternatively, an attenuated IFN-β-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 CD25 Treg cells. In addition, subsets of dendritic cells
(DCs) also express CD4 and direct therapeutic effects of IFN-β 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 9
CD4 Antibodies
Ab Clones Patent Assignee Comments
Non-human primate
CE9.1 US 7,452,534 Biogen Idec
variable regions
TRX1 US 7,541,443 Tolerrx Humanized
1E11, 1G2, 6G5,
US 8,231,877 GenPharm Human
10C5, 4D1
[0205] A role for CD8 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-β on CD8+ T cells in
9293452-1
MS patients (Zafranskaya, 2006 Immunol 121:29). Therefore, directing an attenuated IFN-
β directly to CD8 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 10
CD8 Antibodies
Ab Clones Patent Assignee Comments
Hybridomas
Ortho-McNeil deposited at ATCC
37B1, 8G6 US 7,247,474
Pharmaceutical, Inc. (HB-12441, HB-
12657)
Ortho Hybridoma
OKT8 US 4,361,550 Pharmaceutical deposited at ATCC
Corporation (CRL-8014)
Baylor Research
Several examples US2009/0304659
Institute
[0206] 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 IFNβ.
Examples antibodies that could be used in the present invention include the following:
CD71 antibodies include BA120g (US 7736647) and various antibodies mentioned in
Wang et al (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 (US 7,700,740). An example of an antibody to
CD86 includes 1G10H6D10 (US 6,071,519). HLA-DR antibodies include HD3, HD4,
HD6, HD7, HD8 and HD10 (US 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.
9293452-1
Examples of antibodies to ICOS include JMabs (US 6,803,039) and JMab 136
(US2011/0243929).
Further of these examples of antibodies to these targets are shown in the Tables
11 and 12
Table 11
CD25 Antibodies
Ab Clones Patent Assignee Comments
‘Anti-tac’ Abs US 5,530,101 PDL, Inc daclizumab
Chimeric, inhibits
RFT5 US 6,521,230 Novartis AG
Human antibodies,
AB1, AB7, AB11, US 8,182,812 (or prevent CD25-IL-2
Genmab A/S
AB12 US 7,438,907) interaction and
inhibit MLR
Table 12
CD44 Antibodies
Ab Clones Patent Assignee Comments
H90 US2007/0237761 Chimeric
1A9, 2D1, 14G9,
US2010/0092484 Human
10C8
Binds CD44
SACK-1 US 7,816,500 Sackstein
glycoforms
In another embodiment of the invention, an attenuated version of IFN-β 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-
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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-β 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 (US 7,557,189), any of 14
antibodies described in US patent application US2012/0082670, or the antibody known as
huM195 (US5693761). Antibodies to CD115 that could be used include Ab1 and Ab16
(US 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 (US 7,998,738, ATCC deposit
number PTA-11614). The attenuated IFN-β could alternatively be directed to the CD14
antigen, present primarily on macrophages. Examples of CD14 antibodies are shown in
Table 13.
Table 13
CD14 Antibodies
Ab Clones Patent Assignee Comments
4C1 US 6,245,897 Seikagaku Corporation Mouse Ab
Mochida Pharmaceutical Humanized, inhibits
F10243 US 7,264,967
Co. CD14/TLR binding
US 8,252,905/ Part of fusion
F1024, F1031- Mochida Pharmaceutical
US proteins with
13-2 Co.
2008/0286290 protease
[0210] In yet another embodiment, targeting CD52-expressing cells would deliver IFN-β
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-β to the key cell types known to be directly
affected by IFN-β and would facilitate its therapeutic activity in MS. Examples of CD52
9293452-1
antibodies that could be used according to the present invention include, but are not limited
to DIVHv5/DIVKv2 (US 7,910,104), any of the CD52 antibodies disclosed in
(US2012/0100152) or CAMPATH.
Any of the above mentioned, antibody-targeted attenuated IFNβ 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.
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 Behçet's disease and chronic uveitic macular edema and other types of uveitis,
since IFNa has been shown to render therapeutic benefit (Deuter, Dev Ophthalmol. 51:90-
7. 2012)
[0213] Examples of inflammatory disease conditions contemplated by the present
disclosure include but are not limited to those disease and disorders which result in a
9293452-1
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.
[0214] The sequence of human interferon-b1 is shown below: (SEQ ID NO: 191)
* * * *
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
Using the numbering scheme above (residues 1-166), known mutations (at
positions indicated by asterisks) in human IFNb that reduce its activity include those listed
in Table 14.
Table 14. IFN b bbb 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
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*based on anti-proliferation activity
**the C17S mutation was made in order to remove the unpaired cysteine in the native
sequence of IFNb 1
[0216] References:
(1) Runkel, L., Pfeffer, L., Lewerenz, M., Mogensen, K. (1998). Differences
in Activity between a and b Type I Interferons Explored by Mutational
Analysis. J. Biol. Chem. 273: 8003-8008
(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.
(3) In-house results
In still another embodiment of the present invention, the IFN is IFN-l
( A2), which may be used for any of the applications described more
thoroughly for IFNa or IFNb .
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
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corresponding tumor associated antigens. It will also be appreciated that type I interferons
can be combined with other agents to further improve their effectiveness.
Type I interferons can also display anti-viral properties. IFNa 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-α 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).
In one embodiment, IFN-α 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
9293452-1
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 November 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-α would target IFN-α activity to PS expressing virus-infected cells without displaying
the systemic safety issues related to IFN-α. 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.
It should be understood that antibody-targeted attenuated IFNl could also be used
in much the same way as IFNa for the targeting virally infected cells (S. V. Kotenko, G.
Gallagher, V. V. Baurin et al., “IFN-λs mediate antiviral protection through a distinct class
II cytokine receptor complex,” Nature Immunology, vol. 4, no. 1, pp. 69–77, 2003).
In one embodiment Type II IFNs, namely INFγ, may also be attenuated and
attached to antibodies that direct them to specific cell types. IFNγ 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 IFNγ 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-IFNγ fusion proteins in mammalian cells and showed that both the
antibody and the IFN were functional. Alternatively, a single-chain dimer version of IFNγ,
as described by Lander (J Mol Biol. 2000 May 26; 299(1):169-79) may be used in the
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fusion protein. In addition to IFNγ’s anti-proliferative effect on the targeted tumor cells, it
may also have another effect specifically on breast cancer cells: IFNγ has been shown to
restore antiestrogen sensitivity to breast cancer cells (Mol Cancer Ther. 2010 May; 9(5):
1274–1285) and so an attenuated-IFNγ attached to a breast cancer antigen antibody may be
therapeutically useful in combination with antiestrogen therapy. By attenuating IFNγ via
mutation, a more cancer-selective form of IFNγ may be produced. Two attenuating
mutations in IFNγ 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 ~18-fold
reduced affinity for the IFNγ receptor compared to wild type IFNγ, and had a ~100-fold
reduced antiviral activity compared to wild type IFNγ. The des-(N25, G26) variant had a
~140-fold reduced affinity for the IFNγ receptor compared to wild type IFNγ, and had a
~10-fold reduced antiviral activity compared to wild type IFNγ. Examples of fusion
proteins comprising antibodies to tumor cell surface targets and attenuated mutants of IFNg
include the following: Rituximab may be used as fusion protein with one of these
attenuated IFNg using a 7 amino acid linker described by Sharifi to produce the fusion
protein construct “Rituximab-HC-L7-IFNg (D [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 IFNγ 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 (~50x reduced affinity), D21K (~100x reduced affinity), A23Q
(~2,500-fold reduced binding), A23V (~2,000-fold reduced binding) and D24A (~4-fold
reduced binding). These attenuated IFNg may be used as fusions in combination with anti-
CD38 antibodies, to generate the fusion protein construct “X355/02-HC-L7-IFNg (S20I)
IgG1” (composed of SEQID NOS:380 (heavy chain) and 226 (light chain)) or “R10A2-
HC-L7-IFNg (D21K) IgG1” (composed of SEQ ID NOS:382 (heavy chain) and 270 (light
chain)). Other attenuating mutations in IFNγ that may be exploited for the current
invention were described by Fish (Drug Des Deliv. 1988 Feb; 2(3):191-206.)
[0223] Targeted attenuated IFNγ 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,
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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.
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).
CD14 monocytes from peripheral blood are able to differentiate into fibrocytes,
the precursors of fibroblasts, and this process is inhibited by interferon-γ (IFN-γ). A direct
effect of IFN-γ on monocytes was demonstrated in in vitro differentiation studies,
supporting the strategy of targeting an attenuated form of IFN-γ to CD14 monocytes for
the treatment of fibrotic disease (Shao, 2008 J Leukoc Biol 83:1323).
Experimental evidence exists that IFN-γ 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-γ 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-γ through inhalation of an
aerosol form (Diaz, 2012 J Aerosol Med Pulm Drug Deliv 25:79), such that the lungs may
achieve sufficient IFN-γ activity to produce benefit at an overall safe systemic dose.
Delivering IFN-γ 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-γ 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
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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-
α and –β; 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 15
FAP Antibodies
Ab Clones Patent Assignee Comments
Boehringer
MFP5, BIBH1 US2009/0304718 Ingelheim USA Humanized
Corporation
Many US2012/0128591 Bacac et al. Humanized
Boehringer
F19 US2003/0143229 Ingelheim
International GmbH
9293452-1
Table 16
PDGFR-α and -β Antibodies
Ab Clones Patent Assignee Comments
2.175.3, 2.499.1, Human abs against
US 7,754,859 AstraZeneca AB
2.998.2 PDGFRα
Human abs against
IMC-3G3 US2012/0027767 Imclone LLC
PDGFRα
Human abs against
2C5 US2012/00221267 Imclone LLC
PDGFRβ
In a preclinical model of liver fibrosis, IFN-γ was delivered to hepatic stellate
cells, the equivalent of fibroblasts and responsible for secreting collagen in liver fibrosis,
through liposomes targeting PDGFR-β, thereby enhancing the anti-fibrotic effects of IFN-γ
(Li, 2012 J Control Release 159:261). These data support the concept and the potential
therapeutic benefit gained by delivering IFN-γ activity directly to fibroblasts in fibrotic
diseases, including IPF and liver fibrosis, and validate PDGFR-β as a target for this
approach.
The present invention also contemplates the attenuation and antibody-based
targeting of type III IFNs, including IFNλ1 (IL29), IFNλ2 (IL28A), and IFNλ3 (IL28B) (S.
V. Kotenko, G. Gallagher, V. V. Baurin et al., “IFN-λs 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 IFNλR1 chain (also known as IL28Rα) 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,
9293452-1
11 pages, 2011]). IFNλRs are expressed on most cell types and mediate similar signalling
pathways as the type I IFNs. The antiviral activity of l 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 λ 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-λ), 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 α and λ 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 IFNl 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 IFNl
towards virally infected cells, using for example the targeting antibodies describe above for
the targeting of an attenuated form of IFNa . Mutated, attenuated forms of an IFNl could
also be used to target cancer cells, as described in more detail for IFNa , above.
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).
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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-γ, as was demonstrated in ILtreated Crohn’s disease patients (Tilg,
2002 Gut 50:191).
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).
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-β (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-
γ-targeting, could deliver attenuated IL-10 directly to fibroblasts.
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
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(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).
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).
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).
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 (US 7,915,391) and antibodies DSM ACC 2007,
2008 and 2009 (US 5,545,533). Other antigens for targeting of an attenuated EPO include
CD34, CD45RO, CD45RA, CD115, CD168, CD235, CD236, CD237, CD238, CD239 and
CD240.
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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).
Another example is transforming growth factor β (TGF-β) which is a critical
factor in the regulation of T cell-mediated immune responses and the induction of immune
tolerance. TGF-β knockout mice die from multifocal inflammation and autoimmune
disorders, suggesting an immunosuppressive effect (Shull, 1992 Nature 359:693).
However, TGF-β 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).
In the presence of TGF-β, CD4 CD25 naïve 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-β may help dampen these responses through its
effect on Treg differentiation (Dooley, 2012 Cell Tissue Res 347:245). Similarly, TGF-β
directed to naïve T cells in inflammatory bowel disease could lead to control and
suppression of inflammation (Feagins, 2010 Inflamm Bowel Dis 16:1963).
[0241] Targeting TGF-β specifically to CD4 T cells may leverage the anti-inflammatory
potential of TGF-β, while minimizing its pro-fibrotic properties, and could provide a novel
strategy to combat autoimmune disorders. Alternatively, TGF-β could be targeted soley to
activated T cells using a T cell activation marker, as described above for the discussion of
IFNβ 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.
9293452-1
Another example is Interleukin-4 (IL-4) which is a cytokine that induces the
differentiation of naïve 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.
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).
Diversion towards Th2 may provide a therapeutic benefit in certain types of
diseases. Delivery of IL-4 to CD4 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).
[0245] 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 17
3 -1
IL4 Variant K X 10 S EC T cell proliferation (nM)
off 50
IL4 2.1 0.12
I5R 8.7
T6D 15
E9Q 270 3.1
R81E 6.1
K84D 9.3
9293452-1
3 -1
IL4 Variant K X 10 S EC T cell proliferation (nM)
off 50
R88Q 140 2.5
R88A 760 8.1
N89R 6.1
W91D 8.5
ND. No specific binding found.
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.
[0247] 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).
IL-6, by skewing the terminal differentiation of naïve CD4 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.
[0249] 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).
[0250] 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
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direct link between surviving Tregs and efficacy of adoptive transfer therapy strongly
supports these clinical observations (Baba, 2012 Blood 120:2417).
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).
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 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.
Attenuated mutants of IL-6 include those listed in Table 18.
Table 18
IL6 Variant Binding (% of wild type) EC in XG-1 growth
stimulation assay
(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
These IL-6 mutants and their properties were described by Kalai M et.al. Blood.
1997 Feb 15;89(4):1319-1333
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.
9293452-1
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).
HGF has been shown to alter the fate of epithelial cells and reduce epithelial-
mesenchymal transition (EMT) through its intereference with TGF-β signaling,
antagonizing the process of fibroblastogenesis (Shukla, 2009 Am J Respir Cell Mol Biol
40:643). After organ injury, TGF-β drives conversion of HGF-producing fibroblasts into
collagen-producing myofibroblasts, while HGF in turn inhibits TGF-β 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).
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).
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).
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
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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.
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).
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.
[0263] 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).
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).
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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/Tiα or HTI56 (McElroy, 2004 Eur Respir J 24:664).
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 19
VEGFR Antibodies
Ab Clones Patent Assignee Comments
Shanghai Aosaiersi Human anti-VEGFR2
AC88 US 8,128,932
Biotech Co., Ltd mAb
Antibody 1,
US2012/0058126 Imclone LLC Anti-VEGFR3 Abs
Antibody 2
Korea Research
Institute of
6A6 US2011/0065176 Human, anti-VEGFR
BioScience and
BioTechnology
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 TNFα, TRAIL, Fas Ligand, IFNβ,
IFNγ or IFNλ, which can be targeted to various tumor cell surface antigens as discussed
above for INFα.
9293452-1
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.
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.
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
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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.
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.
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.
[0273] 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.
[0274] Pharmaceutical excipients and additives useful in the present composition include
but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g.,
9293452-1
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.
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.
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.
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-β-cyclodextrin), polyethylene glycols,
flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents,
surfactants (e.g., polysorbates such as “TWEEN® 20” and “TWEEN® 80”), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
[0278] 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.,
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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.
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.
[0280] 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.
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.
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-IFNa fusion protein constructs
Expression vectors:
The DNA encoding the rituximab (Anderson et al., US Patent 5,843,439, Dec. 1,
1998) and palivizumab (Johnson, US Patent 5,824,307, Oct. 20, 1998) variable regions
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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.
(US Patent 7829673) and by Daelken et al. (), respectively, and
subjected to be synthesized by Integrated DNA Technology, Inc. (Coralville, IA) after the
sequence modification to eliminate rare codons and unprefered restriction sites.
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, CA) and Mouse Ig-Primer Sets (Novagen/EMD Chemicals,
San Diego, CA). The sequence determination and sub-cloning of the newly isolated anti-
CD38 antibodies is described in the following sections.
The DNA encoding human interferon-2 a b (IFN2 a b; 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-b 1 (IFNb 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, IA) or GenScript USA Inc. (Piscataway,
NJ) 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.
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 PO1857), a human IgG4 heavy chain constant region
(such as Swissprot accession number P01861 incorporating substitution S228P), human Ig
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kappa constant region (Swissprot accession number P01834) or human Ig lambda constant
region (Swissprot accession number P0CG05) 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:
DNA plasmids encoding the IgGs and IgG-cytokine fusion protein constructs
were prepared using Plasmid Plus Maxi kit (Qiagen, Valencia, CA) 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, CA)
using a commercially available transfection reagent and OptiMEM medium (Invitrogen,
Carlsbad, CA). After allowing for expression for 6 days in an incubator supplied with 5%
CO and gentle shaking, the culture media was isolated and subjected to IgG affinity
purification using Protein G-agarose beads (GE Healthcare, Piscataway, NJ). 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, MA), followed by protein concentration determination using a
NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA).
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 IFNa , were produce at at least 100 mg/L of media, showed high solubility and did not
aggregate as determined by size exclusion chromatography.
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].
Thus for example the construct “Rituximab-HC-L6-IFNa (A145G) IgG1” is the
antibody rituximab, with IFNa 2b (with the A145G point mutation), linked to the C-
terminus of the IgG1 heavy chain, with an intervening linker L6.
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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)
L6: SGGGGS (SEQ ID NO:132)
L16: SGGGGSGGGGSGGGGS (SEQ ID NO:133)
Method for measuring antigen-targeted activity of antibody-IFNa fusion protein
constructs
“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®, Cat #G7570, from Promega (Madison,
Wisconsin). 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:
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 x 10
and 0.8 x 10 viable cells/ml in RPMI 1640 (Mediatech, Inc., Manassas, VA, cat # 10
CV) with 10% Fetal Bovine Serum (FBS; Hyclone, Logan, UT cat# SH30070.03). Cells
were harvested by centrifuging at 400g 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 x 10 cells/ml in RPMI 1640 + 10% FBS. Then, 50 m 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, NY cat# 3879), test articles were serially
diluted in duplicate in RPMI 1640 + 10% FBS. Then, 50 m l/well was transferred from the
dilution plate to the experimental plate. The experimental plate was then incubated for four
days at 37 C with5% CO .
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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 m l/well. The plate was shaken for two minutes.
Then, 100 m l/well was transferred from the experimental plate to a 96 well flat bottom
white opaque plate (hereafter, “assay plate”; BD Biosciences, Franklin Lakes, NJ 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, MA, model# 1420-041) on the luminometry channel and the
luminescence was measured. Results are presented as “relative luminescence units
(RLU)”.
Data was analyzed using Prism 5 (Graphpad, San Diego, CA) 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 IFN2 a b (or some other form of IFN with a
known potency relative to IFNa 2b) was calculated as a ratio of EC50s.
[0296] 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.
“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 ) 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
5 4
density of 4.0 x 10 to 6.0 x 10 cells/ml. ARP-1 concentration was adjusted to 1.0 x 10
cells/ml prior to assay.
Method for measuring non-antigen-targeted activity of antibody-IFNa fusion protein
constructs
“Off-target assay” (also sometimes referred to herein as the “not-targeted” assay):
The iLite assay from PBL Interferon Source (Piscataway, NJ,Cat# 51100), was performed
largely as described by the manufacturer with the addition of a human IgG blocking step.
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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°C
freezer and allowed to equilibrate to room temperature. Then, 50 m l of the diluent was
added per well to the assay plate. The vial of manufacturer-supplied reporter cells was
removed from the -80°C freezer and thawed in a 37 C water bath. Then, 25 m l aliquiots of
cells were dispensed into each well of the assay plate. Next, 12.5 m l of 8 mg/ml human
IgG that was diluted into RPMI 1640 + 10% FBS (Sigma Chemicals, St. Louis, MO; cat#
I4506) was added per well. The contents were mixed and incubated at 37 C for 15
minutes. On a separate “dilution plate,” test articles were serially diluted in duplicate in
RPMI 1640 + 10% FBS. Then, 12.5 m l of the test articles were transferred from the
dilution plate to the assay plate. The assay plate was then incubated at 37 C with 5% CO
for 17 hours. The manufacturer-supplied assay buffer and substrate were removed from
the -80 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 m 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).
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Results: Antigen-specificity of antibody-IFNa fusion protein constructs
Figure 6 shows the interferon activity of free IFNa 2b (SEQ ID NO:3; “IFNa ” in
figure) as well as IFNa 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 IFN2 a b-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-IFNa fusion
protein constructs”and is hereafter abbreviated as the “off-target assay.” “Rituximab-HC-
L6-IFN a 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 IFNa 2b (SEQ ID NO:3); this heavy chain-
linker-IFNa sequence is shown as SEQ ID NO:280. “Isotype-HC-L6-IFN a 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 IFNa 2b (SEQ ID NO:3); this heavy chain-linker-IFN2 a b sequence is
shown as SEQ ID NO:294. In this assay, free IFNa 2b showed an EC for activating gene
expression through an interferon response element (IRE) of 1.9 pM. By attaching IFNa 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 IFNa 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 IFNa 2b caused a modest (3-4x) decrease in its non-antigen-targeted IFN
activity. This is consistent with what has been reported by other (for example in US
7,456,257). Neither Palivizumab nor Rituximab alone (without the fusion to an interferon)
showed any activity in this assay (data not shown).
To determine whether the antibody-IFN2 a b fusion protein constructs had
enhanced activity relative to free IFN2 a b on cells that do display the corresponding
antigen on their cell surface, their effect on Daudi cells, which display the CD20 antigen of
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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-IFNa 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-IFNa 2b fusion protein construct (Rituximab-
HC-L6-IFN a IgG1) was 3.25-fold (1.3/0.4 = 3.25) more potent than free IFNa 2b (Figure
7). In other words, the attachment of Rituximab to IFNa 2b resulted in slightly reduced
(3.1-fold) activity towards antigen-negative cells (Figure 6) but slightly increased (3.25-
fold) activity towards antigen-positive cells (Figure 7). Overall, the antibody attachment
therefore increased the antigen-specificity index (ASI), defined as the fold increased
potency relative to free IFN2 a b on antigen-positive cells multiplied by the fold decreased
potency relative to free IFN2 a b on antigen-negative cells, by 10-fold (3.1 x 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.
The results presented in Figure 8 are consistent with antibody-based targeting
relying on antibody-antigen reactivity: the Rituximab-IFNa fusion protein construct
(Rituximab-HC-L6-IFN-a IgG1) was 12-fold (2.2/0.18 = 12) more potent in reducing
viability of the CD20 Daudi cells than the Palivizumab-IFNa fusion protein construct
(Isotype-HC-L6-IFN-a IgG1), the antigen for which is not present on the Daudi cells.
The modest reduction in IFNa activity that occurred as a result of linking it to an
antibody may not be sufficient to prevent the toxicity of the IFNa component of the
construct in human subjects. Various mutations were therefore introduced into IFN2 a b in
order to reduce its activity and toxicity. For example, five different mutant versions of
IFNa 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-IFNa IgG1 (as also used in the experiments shown in
Figures 6-8). The five mutant versions were R144A, A145G, R33A+YNS, R33A and
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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.
[0303] Figures 9, 10 and Table 20 show the degree of reduced interferon activity for each
of these Rituximab-attenuated IFNa 2b fusion protein constructs relative to free, wild type
IFNa 2b, on antigen-negative (i.e. CD20-negative) cells. The R144A mutant of the
Rituximab-IFNa 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. Figure 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-IFNa IgG1); since Rituximab-
HC-L6-IFNa IgG1 is 3.8-fold less potent on antigen negative cells than free, wild type
IFNa 2b (data from Figure 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 IFNa (303 x 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.
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Table 20
Fusion protein Targeted Potency Relative Non-Targeted Potency Antigen-Specificity
construct Test Index (ASI;
to free IFNa 2b (EC50 Relative to free IFNa 2b
Article calculated as Column
IFNa 2b/EC50 Fusion (EC50 IFNa 2b/EC50
A/Column B)
protein construct) Fusion protein construct)
Column A Column B
3.6 0.26 14
Ritux-IFNa 2b
0.86 0.0026 330
Ritux-IFNa 2b
(R144A)
1.2 0.0020 600
Ritux-IFNa 2b
(A145G)
1.6 0.00093 1,700
Ritux-IFNa 2b
(R33A+YNS)
Ritux-IFNa 2b 0.0022* No detectable activity in ND
non-targeted assay
(R33A)
0.23* 0.00086* 270
Ritux-IFNa 2b
(R144A+YNS)
* Free IFNa 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-
IFNa IgG1, which was assayed on the same day and same plate, multiplied by a correction
factor based on the relative activity of IFNa 2b vs Rituximab-HC-L6-IFNa IgG1 (i.e. data
shown in the second row from the top) measured on a different day.
Surprisingly, when the amount of interferon activity of these highly attenuated
rituxumab-mutant IFN2 a b fusion protein constructs was measured on antigen-positive
cells (Daudi, CD20 ), there was generally very little attenuation compared to the wild type
IFNa 2b version of the Rituximab-IFNa 2b fusion protein construct (Figures 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-IFNa 2b wild type construct on the antigen-positive (Daudi)
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cells. This was in contrast to the 277-fold (6100/22 = 277; Figure 9) reduced activity on
antigen-negative cells. The mutations in the IFNa 2b, in the context of the Rituximab-
IFNa 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-
IFNa 2b (R33A+YNS) IgG1 fusion protein construct exhibited a substantially greater
antigen-specificity index (ASI, 1,700-fold) compared to Rituximab-HC-L6-IFNa 2b IgG1
(10- to 14-fold) or free IFNa 2b (1-fold, by definition), suggesting that its off-target effects
in vivo will be substantially reduced.
Other Rituximab-IFN2 a b constructs with mutations in the IFN2 a b portion also
showed surprisingly little reduced activity on antigen-positive cells (Figures 11 and 12)
relative to their reduced potency on antigen-negative cells (Figures 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 IFNa 2b on antigen-negative cells, but showed 0.23 – 1.2-fold of the potency
of wild type IFNa 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
(Figure 10). These results are summarized in Table 20.
To determine whether this dramatic difference in the ability of the mutations in
the IFNα 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 IFNa and characterized. Some of these experiments were performed using the
antibody G005 (De Weers et al. (US Patent 7829673)); the sequences of the heavy and
light chains for this human antibody are shown as SEQ ID NOS:135 and 134, respectively.
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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
[0308] The extracellular domains (ECD) of human and cynomolgus monkey CD38
proteins were each formatted to include a cleavable N-terminal leader sequence, an
Avitag™, 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
[0309] 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
HEK293-6E cells were cultured in complete cell growth media (1 L of F17
medium (Invitrogen ), 9 mL of Pluronic F68 (Invitrogen ), 2mM Glutamine containing
% (w/v) Tryptone NI (Organotechnie ) with Geneticin (50 mg/mL, Invitrogen ) at 50
μl/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 C with 5% CO and 120 rpm without
Geneticin. The next day, 12.5 mL of Tryptone was added along with 250 µl of Geneticin
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per 500 mL culture. The culture was incubated at 37 C, 5% CO and 120 rpm. After 7
days, the supernatant was harvested by centrifugation and was ready for purification.
Expression and purification of antibodies
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 1x 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 1X 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
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™ 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
X PBS (pH 7.4). The absorbance at 280 nm (A ) 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
[0313] The Avitag™ motifs of human and cynomolgus monkey CD38 EDs were
biotinylated according to manufacturer’s directions (Avidity LLC, Aurora, CO). Excess
unconjugated biotin was removed from the biotinylated proteins by desalting into 1x PBS
using a 7KD molecular weight cut off (MWCO) Zeba spin column (Thermo Scientific,
Logan, UT) according to manufacturer’s instructions. Successful biotinylation of CD38
ED proteins was confirmed using a combination of polyacrylamide gel electrophoresis and
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Western blotting. Western blots were probed using Streptavidin-HRP (BD Biosciences,
San Diego, CA) 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
[0314] FAbs that bind to both human and cynomolgus monkey CD38 EDs were isolated
from a naïve phagemid library comprising approximately 2.5 x 10 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 naïve phagemid library that expressed antibody fragments in the Fab format.
[0315] 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).
Each phage display campaign involved three rounds of panning. For each round,
~2.5 x 10 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 m 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.
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 m L of streptavidin-coupled
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Dynabeads (Invitrogen, Carlsbad, CA) were added and the mixture was incubated a further
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
(1x PBS with 0.5% skim milk) or PBS-T wash buffer (1x 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).
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 (2x yeast-tryptone (2YT) growth
media) and allowed to infect the cells during a 30 minute incubation at 37°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 m 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
[0319] 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 m g/mL carbenicillin and 2% glucose) in 96-well deep-well plates
(Costar) and incubated overnight at 37°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 m g/mL carbenicillin) and grown to an optical density (600 nm) of
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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°C for 16 hrs.
FAb samples were prepared by harvesting cells by centrifugation (2,000g, 10
mins) and performing a lysozyme extraction. The cell pellet was resuspended in 200 m L of
lysis buffer (160 m g/mL lysozyme, 10 m g/mL RNase A, 5 m g/mL DNase and complete
protease inhibitors (Roche, Nutley, NJ)) and shaken at 400 rpm for 30 minutes at 21°C.
Following addition of a further 100 m l of lysis buffer, the reactions were incubated for a
further 30 minutes as described previously. Clarified lysates were isolated following
centrifugation at 3,000g for 10 minutess and stored at 4°C until required.
[0321] 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 m 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 (1xPBS supplemented with 0.1%
Tween®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, CA) 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 m L
3,3',5,5'-Tetramethylbenzidine (Sigma-Adrich, St. Louis, MO) and quenching with 50 m 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’.
[0322] 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 Figure 13.
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Table 21
Campaign Number FAb name V sequence V /V sequence
H K L
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
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
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 0JG, UK) in flow cytometry-based
assays was tested. Briefly, viable RPMI-8226 cells (2 x 10 , as judged by trypan blue
exclusion) were incubated with each antibody or with a human IgG isotype control
antibody preparation (Sigma-Aldrich, St. Louis, MO) at various concentrations in 100 μl
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 μl 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, CA) using EV, side scatter
and FL-1 gating. Results are expressed as mean fluorescent intensity (MFI) plotted against
protein concentration (Figure 14).
Generation of Anti-CD38 antibodies by genetic immunization
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.
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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.
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.
Four days later, the rats were sacrificed and their lymphocytes fused with
myeloma cells using polyethylene glycol (HybriMax™; 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
[0328] Duplicate 100 μL samples of each hybridoma supernatant were coated onto
separate wells of a maxisorp ELISA plate (Nunc Plasticware, Thermo Scientific,
Rochester, NY 14625, USA) through incubation at room temperature for an hour. Plates
were washed three times in 1xPBS-T and subsequently blocked by addition of
2%BSA/1xPBS. 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 0.1μg
of biotinylated human CD38 in a final volume of 100 μL 1xPBS. To the second well of
each rat antibody duplicate well was added 0.1μg of biotinylated cynomolgus monkey
CD38 ED in a final volume of 100 μL 1xPBS. Plate wells were washed as described
previously prior to detection of bound biotinylated CD38 ED using a Streptavidin-HRP
conjugate (BD Biosciences, San Diego, CA). Plates were washed as above to remove
unbound Streptavidin-HRP conjugate and the assay signal developed by incubation with
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50 m L 3,3',5,5'-Tetramethylbenzidine (Sigma-Aldrich) and quenching with 50 m L 1 M
HCl. Assay signals were read at A nm using a microplate reader (BMG Labtech, Cary,
NC). 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
Viable RPMI-8226 cells (2 x 10 , as judged by trypan blue exclusion) were
incubated with 100 m L of rat hybridoma supernatant for 20 minutes on ice in the dark.
Cells were washed twice with FACS buffer (1x PBS plus 1% FCS) before incubation for
minutes in 100 μl 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, CA) 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 22
Rat Binding to Human Binding to FACS binding to
antibody CD38 ED (ELISA) Cynomolgus monkey RPMI-8226 cells
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
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Rat Binding to Human Binding to FACS binding to
antibody CD38 ED (ELISA) Cynomolgus monkey RPMI-8226 cells
CD38 ED (ELISA) (MFI)
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
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, CA] SMART RACE kit; Ambion Life Technologies [Foster
City, CA] 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.
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,
CA) according to manufacturer’s instructions. Single colonies from transformations were
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cultured and plasmid DNA prepared using a GenElute™ plasmid miniprep kit (Sigma-
Aldrich, St. Louis, MO). DNA inserts were sequenced and antibody variable regions
identified in the conceptually translated protein sequences.
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 23
Rat antibody Light chain 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
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 µl/min, 20 µl of 5 µg/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 10mM 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 , k and K values. This
d a D
data shows that cross-reactivity for human and cynomolgus monkey CD38 is maintained
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on conversion of the human phage-derived Fabs into human IgGs and rat antibodies into
chimeric rat-human IgGs (Table 24).
Table 24
Antibody CD38 Ligand ka (1/Ms) x10 kd (1/s) K (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
[0334] To determine whether the surprising result obtained with an anti-CD20 antibody
fused to an attenuated IFNa 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 IFNa (SEQ ID NO:3), with or without various
attenuating mutations was made. Figure 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.
Figure 15 shows the off target activity of free wild type IFNa 2b (IFNa ) (SEQ ID
NO:3) vs. wild type IFNa 2b fused to the C-terminus of the CD38 antibody G005 (De
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Weers et al. (US Patent 7829673). The latter fusion protein construct (G005-HC-L0-IFNa
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 IFNa and is described by SEQ ID NOS:150
(heavy chain) and 134 (light chain). As illustrated in figure 15, the anti CD38 antibody-
non-attenuated IFNα2b fusion protein construct was 27-fold less potent (19.5/0.726 = 27)
than free IFN2 a b in the off-target assay (e.g. in the absence of CD38-targeting). Figure 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-IFNa IgG4 fusion protein construct was
3.6-fold (14.7/4.08 = 3.6) more potent than free IFNa 2b, presumably due to the targeted
delivery of the IFN to the CD38 myeloma cells. Therefore, the G005-HC-L0-IFNa IgG4
fusion protein construct has an antigen specificity index (ASI) of 97 (27 x 3.6 = 97; Table
).
Table 25
Test Article EC50 On EC50 IFNaa EC50 Off EC50 IFNaa Antigen
a a a a
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA (Off
(pM) ARP- iLite Index
(On Target; Target; iLite)
1 Column
ARP-1)
Column 5
IFNa 10.8 1.00 0.260 1.00 1.00
G005-HC-L0- 3.6* 0.037** 97
IFNa IgG4
G005-HC-L0- 186 0.0581 25,800 1.01 x 10 5,750
IFNa (R144A)
IgG4
-6
G005-HC-L0- 290 0.0372 1.08 x 10 2.41 x 10 15,400
IFNa (R144S)
IgG4
G005-HC-L0- ND ND >10 ND ND
IFNa (R144E)
IgG4
G005-HC-L0- ND ND 9,970 2.61 x 10 ND
IFNa (R144G)
IgG4
G005-HC-L0- ND ND 1,690 1.54 x 10 ND
IFNa (R144H)
IgG4
G005-HC-L0- ND ND <100 ND ND
IFNa (R144K)
IgG4
9293452-1
Test Article EC50 On EC50 IFNaa EC50 Off EC50 IFNaa Antigen
a a a a
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA (Off
(pM) ARP- iLite Index
(On Target; Target; iLite)
Column
1 ARP-1)
Column 5
G005-HC-L0- ND ND 431 0.000603 ND
IFNa (R144N)
IgG4
G005-HC-L0- ND ND 3,500 7.43 x 10 ND
IFNa (R144Q)
IgG4
G005-HC-L0- 333 0.0324 30,800 8.44 x 10 3,840
IFNa (R144T)
IgG4
G005-HC-L0- 306 0.0353 92,100 2.82 x 10 12,500
IFNa (R144Y)
IgG4
-6
G005-HC-L0- 257 0.0420 1.59 x 10 1.64 x 10 25,600
IFNa (R144I)
IgG4
G005-HC-L0- 191 0.0565 26,700 9.74 x 10 5,800
IFNa (R144L)
IgG4
G005-HC-L0- ND ND 86,900 2.99 x 10 ND
IFNa (R144V)
IgG4
G005-HC-L0- 23.8 0.454 2,040 0.000127 3,570
IFNa (A145G)
IgG4
G005-HC-L0- 222 0.0486 52,600 4.94 x 10 9,840
IFNa (A145D)
IgG4
G005-HC-L0- ND ND <100 ND ND
IFNa (A145E)
IgG4
G005-HC-L0- 113 0.0956 24,900 1.04 x 10 9,190
IFNa (A145H)
IgG4
G005-HC-L0- ND ND 28.9 0.00900 ND
IFNa (A145I)
IgG4
-7 5
G005-HC-L0- 174 0.0621 6.62 x 10 3.93 x 10 1.58 x 10
IFNa (A145K)
IgG4
G005-HC-L0- ND ND 239 0.00109 ND
IFNa (A145L)
IgG4
G005-HC-L0- ND ND 309 0.000841 ND
IFNa (A145N)
IgG4
9293452-1
Test Article EC50 On EC50 IFNaa EC50 Off EC50 IFNaa Antigen
a a a a
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA (Off
(pM) ARP- iLite Index
(On Target; Target; iLite)
Column
1 ARP-1)
Column 5
G005-HC-L0- ND ND 709 0.000367 ND
IFNa (A145Q)
IgG4
G005-HC-L0- ND ND >10 ND ND
IFNa (A145R)
IgG4
G005-HC-L0- ND ND <2 ND ND
IFNa (A145T)
IgG4
G005-HC-L0- 91.4 0.118 19,200 1.35 x 10 8,740
IFNa (A145Y)
IgG4
In order to determine whether the ASI could be increased, as was observed for the
anti-CD20-IFNa fusion protein constructs, several variants were constructed by
attenuating the IFN portion of the anti-CD38-IFNa 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 Figures
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 Figures 17-38).
Table 26 characterizes the CD38 antibody G005, fused in various configurations
via the C-terminus of the heavy chain to IFNa 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
IFNa ), but substantially maintained the potency exhibited by free IFNa on CD38 positive
cells (Daudi). The G005-HC-L0-IFNa (R144A) IgG4 construct (composed of SEQ ID
NOS:152 (heavy chain) and 134 (light chain)), for example, has a 10 -fold lower potency
9293452-1
than free, wild type IFNa on antigen negative cells but its potency is reduced only 3.5-fold
(2.7/0.77 = 3.5) vs. free, wild type IFNa on antigen positive cells (Table 26). This gives an
Antigen Specificity Index (ASI) of 29,000 for this fusion protein construct.
Table 26
Test Article (TA) EC50 On EC50 Off Antigen
EC50 IFNa EC50 IFNa
Target /EC50 TA Target (pM) /EC50 TA Specificity
(pM) iLite Index
(On Target; (Off Target;
Daudi Column 3/
Daudi) iLite)
Column 5
IFNa 0.77 1.0 0.30 1.0 1.0
G005-HC-L6- 1.3 0.59 11,000 2.7 x 10 22,000
IFNa (R144A)
IgG4
G005-HC-L0- 2.7 0.29 30,000 1.0 x 10 29,000
IFNa (R144A)
IgG4
G005-HC-L6- 1.9 0.41 2,600 0.00012 3,400
IFNa (R144A)
IgG1
G005-HC-L0- 7.3 0.11 6,800 4.4 x 10 2,500
IFNa (R144A)
IgG1
[0338] Table 27 shows examples using another IFNa 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-IFNa (A145G) IgG4
construct (composed of SEQ ID NOS:148 (heavy chain) and 134 (light chain)), for
example, showed an ASI of 20,000.
9293452-1
Table 27
Test Article (TA) EC50 On EC50 Off Antigen
EC50 IFN a aaa EC50 IFN a aaa
Target Target (pM) Specificity
/EC50 TA /EC50 TA
(pM) iLite Index
(On (Off
Daudi Target; Target; Column
Daudi) iLite)
Column 5
0.48 1.0 0.087 1.0 1.0
IFNa
G005-HC-L0- 0.74 0.65 510 0.00017 3,800
IFNa (A145G)
IgG1
G005-HC-L6- 1.0 0.48 730 0.00012 4,000
IFNa (A145G)
IgG1
G005-HC-L6- 0.59 0.81 2200 4.0 x 10 20,000
IFNa (A145G)
IgG4
Table 28 shows examples in which the mutated IFNa 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
,900 and 7,200, respectively.
Table 28
Test Article EC50 On EC50 Off Antigen
EC50 IFN a aaa EC50 IFN a aaa
(TA) Target (pM) Target (pM) Specificity
/EC50 TA /EC50 TA
Daudi iLite Index
(On (Off Target;
Column
Target; iLite)
Daudi)
Column 5
1.1 1.0 0.21 1.0 1.0
IFNa
G005-LC-L6- 8.5 0.13 12,000 1.8 x 10 7,200
IFNa (A145G)
IgG1
G005-LC-L0- 21 0.052 24,000 8.8 x 10 5,900
IFNa (A145G)
IgG1
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 cells.
Using this method, the ASIs for these fusion protein constructs ranged from 1,200-55,000.
9293452-1
Table 29
Test Article EC50 On EC50 Off Antigen
EC50 IFN a aaa EC50 IFN a aaa
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA (Off
(pM) ARP- (On Target; iLite Target; iLite) Index
1 Column 3/
ARP-1)
Column 5
6.0 1.0 0.30 1.0 1.0
IFNa
G005-HC-L6- 28 0.21 11,000 2.7 x 10 7,800
IFNa (R144A)
IgG4
G005-HC-L0- 85 0.071 30,000 1.0 x 10 7,100
IFNa (R144A)
IgG4
G005-HC-L6- 21 0.29 2,600 0.00012 2,400
IFNa (R144A)
IgG1
G005-HC-L0- 110 0.054 6,800 4.4 x 10 1,200
IFNa (R144A)
IgG1
Table 30
Test Article EC50 On EC50 Off Antigen
EC50 IFNa EC50 IFNa
(TA) Target Target Specificity
/EC50 TA /EC50 TA (Off
(pM) ARP- (pM) iLite Index
(On Target; Target; iLite)
1 ARP-1) Column 3/
Column 5
9.5 1.0 0.087 1.0 1.0
IFNa
G005-HC-L0- 8.0 1.2 510 0.00017 7,100
IFNa (A145G)
IgG1
G005-HC-L6- 4.0 2.4 730 0.00012 20,000
IFNa (A145G)
IgG1
G005-HC-L6- 4.4 2.2 2200 4.0 x 10 55,000
IFNa (A145G)
IgG4
9293452-1
Numerous other examples of mutated versions of IFNa , in the context of the
G005-HC-L0-IFNa 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 IFNa activity compared to free wild type IFNa In this context, the A145V
and A145S mutants did not show appreciable attenuation. Any of these point mutated,
attenuated versions of IFNa 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 IFNa 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)).
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 IFNa . The
A145E mutation had minimal impact on the IFNa activity, however, the A145D mutation
drastically reduced activity (by 20,000-fold) and resulted in a construct with high ASI
(9,840).
[0343] Other examples of anti-CD38 antibody-attenuated IFNa fusion protein constructs
are shown in Tables 31-33. In addition to the G005 antibody constructs, these tables show
9293452-1
the on-target activity, the off-target activity, and the ASI for anti-CD38 antibody-
attenuated IFNa fusion protein constructs based on certain novel antibodies. Fusion
protein constructs of these antibodies with the IFNa A145D or R144A mutations include
the following:
[0344] X910/12-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:248 (heavy
chain) and SEQ ID NO:242 (light chain)
X910/12-HC-L0 IFNa (R144A) IgG4 composed of SEQ ID NOS:246 (heavy
chain) and SEQ ID NO:242 (light chain)
; X913/15-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:256 (heavy
chain) and SEQ ID NO:250 (light chain));
X913/15-HC-L0 IFNa (R144A) IgG4 composed of SEQ ID NOS: 254 (heavy
chain) and SEQ ID NO:250 (light chain))
X355/02-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:232 (heavy
chain) and SEQ ID NO:226 (light chain);
[0349] X355/02-HC-L0 IFNa (R144A) IgG4 composed of SEQ ID NOS:230 and SEQ
ID NO:226 (light chain);
X355/07-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:240 (heavy
chain) and SEQ ID NO:234 (light chain);
X355/07-HC-L0 IFNa (R144A) IgG4 composed of SEQ ID NOS:238 and SEQ
ID NO:234 (light chain);
R5D1-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:262 (heavy chain)
and 258 (light chain);
R5E8-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:268 (heavy chain)
and 264 (light chain); and
[0354] R10A2-HC-L0 IFNa (A145D) IgG4 composed of SEQ ID NOS:274 (heavy
chain) and 270 (light chain).
9293452-1
These fusion protein constructs all showed high ASIs, ranging from 3,820
(X910/12-HC-L0-IFNa (R144A) IgG4) to 166,000 (X355/02-HC-L0-IFN a (A145D)
IgG4).
Table 31
Test Article EC50 On EC50 IFNa EC50 Off EC50 IFNa Antigen
(TA) Target Target Specificity
/EC50 TA /EC50 TA (Off
(pM) (pM) iLite Index
(On Target; Target; iLite)
ARP-1 Column 3/
ARP-1)
Column 5
8.91 1.00 0.499 1.00 1.00
IFNa
G005-HC-L0- 220 0.0405 73,200 6.82 x 10 5,940
IFNa (R144A)
IgG4
X910/12-HC- 191 0.0466 41,000 1.22 x 10 3,820
L0- IFNa
(R144A) IgG4
X913/15-HC- 84.1 0.106 29,600 1.69 x 10 6,270
L0- IFNa
(R144A) IgG4
X355/02-HC- 147 0.0606 70,500 7.08 x 10 8,560
L0- IFNa
(R144A) IgG4
X355/07-HC- 61.2 0.146 70,300 7.10 x 10 20,600
L0- IFNa
(R144A) IgG4
9293452-1
Table 32
Test Article EC50 On EC50 Off Antigen
EC50 IFNa EC50 IFNa
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA
(pM) iLite Index
(On Target; (Off Target;
ARP-1 Column 3/
ARP-1) iLite)
Column 5
43.0 1.00 0.304 1.00 1.00
IFNa
G005-HC-L0- 85.7 0.502 47,100 6.44 x 10 78,000
IFNa (A145D)
IgG4
X910/12-HC- 336 0.128 22,500 1.35 x 10 9480
L0- IFNa
(A145D) IgG4
X913/15-HC- 68.1 0.631 31,800 9.59 x 10 65,800
L0- IFNa
(A145D) IgG4
-6 5
X355/02-HC- 46.0 0.935 53,900 5.64 x 10 1.66 x 10
L0- IFNa
(A145D) IgG4
-6 5
X355/07-HC- 61.9 0.695 61,800 4.92 x 10 1.41 x 10
L0- IFNa
(A145D) IgG4
Table 33
Test Article EC50 On EC50 Off Antigen
EC50 IFNa EC50 IFNa
(TA) Target Target Specificity
/EC50 TA (On /EC50 TA
(pM) Target; ARP-1) (pM) iLite (Off Target; Index
ARP-1 Column 3/
iLite)
Column 5
16.1 1.00 0.271 1.00 1.00
IFNa
R5D1-HC-L0- 51.3 0.314 49,500 5.48 x 10 57,300
IFNa (A145D)
IgG4
R5E8-HC-L0- 89.2 0.180 38,400 7.06 x 10 25,500
IFNa (A145D)
IgG4
R10A2-HC-L0- 32.7 0.492 29,600 9.16 x 10 53,700
IFNa (A145D)
IgG4
X355/02-HC- 104 0.155 81,600 3.32 x 10 46,700
L0- IFNa
(A145D) IgG4
9293452-1
The examples above demonstrate that mutated, attenuated forms of IFNa ,
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 IFNa to two other antigens: CD138 and class I MHC.
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 IFNa 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 Figure 39, this fusion
protein construct, like the anti-CD38-attenuated IFNa 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). Figure
39 shows that a 28pM concentration (4 highest concentration tested) of nBT062-HC-L0-
IFNa (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-IFNa (A145D)
protein.
[0358] 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, CA) and
Mouse Ig-Primer Sets (Novagen/EMD Chemicals, San Diego, CA). 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 IFNa with the A145D mutation
(HB95-HC-L0-IFN a (A145D) IgG4, composed of SEQ ID NOS:316 (heavy chain) and
9293452-1
312 (light chain)) was expressed, and its activity was compared to an isotype control
antibody fused in the same way to the same IFNa mutant (Isotype-HC-L0-IFN a (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 Figure 40a. The class I MHC-targeted
attenuated IFNa is orders of magnitude more potent than the isotype control-attenuated
IFNa fusion protein construct on the same cells, coming within about 9-fold (139/16 = 8.7)
of the wild type IFNa . While HB95-HC-L0-IFNa (A145D) IgG4 shows significant
activity below 100 pM, the isotype-HC-L0-IFNa (A145D) IgG4 shows no significant
activity even at 6 nM.
Figure 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 IFNa fusion protein constructs on the proliferation of ARP-1
cells. Two non-ARP-1 targeted constructs, “Palivizumab-HC-L6-IFNa (A145D) Fab”
(composed of SEQ ID NOS:298 (heavy chain) and 290 (light chain)) and “2D12-HC-L6-
IFNa (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-IFNa (A145D) Fab”
(composed of SEQ ID NOS:320 (heavy chain) and 312 (light chain)), the potency is even
higher than free, wild type IFNa . 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 a aaa
The anti-viral activity of IFNa is well-known and recombinant IFNa is an FDA-
approved treatment for hepatitis C viral infections. The effect of a host cell surface-
targeted vs. non-targeted antibody-attenuated IFNa fusion protein construct on the
cytopathic activity of the EMC virus on A549 cells, which are class I MHC-positive, was
compared.
9293452-1
Methods:
IFN activity was measured using the cytopathic effect inhibition (CPE) assay as
described Rubinstein (J. Virol. 37, 755-8, 1981). Briefly, 10 human adenocarcinoma
A549 cells (ATCC, Manassas, Kansas) per well were incubated with test sample or IFN
(human IFN-a 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 IFNa 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 IFNa 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 IFNa (Intron A, inverted triangles), Anti-
MHC class I targeted attenuated IFNa designated HB95-HC-L0-IFNa (R145D) IgG4
(closed squares), and istoype control (2D12)-attenuated IFNa (Isotype-HC-L0-IFNa
(R145D) IgG4; triangles). Data is plotted as viability vs IFNa molar equivalents.
Results:
Results are shown in Figure 41. In this assay, IFNa 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 IFNa (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 b bbb
IFNb 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 IFNb (SEQ ID NO:91), G005-HC-L0-IFNb IgG4
9293452-1
(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 IFNb
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 IFNb was mutated to a serine in order to improve expression
yields and product homogeneity. Figure 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 IFNb 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
,150–fold (16,100/0.799 =20,150) less potent than free, wild type IFNb . In stark
contrast, Figure 43 shows the potency of these three proteins under conditions in which the
CD38 antibody can target the IFNb to cells is fairly similar. In this assay, the antibody-
attenuated IFNb fusion protein construct (G005-HC-L0-IFNb (R35A) IgG4) is only 1.4–
fold (46.9/32.7 = 1.4) less potent than the antibody-non-attenuated IFNb fusion protein
construct and only 4.5–fold (46.9/10.5 = 4.5) less potent than free, wild type IFNb . 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 IFNa
(Table 20), also holds true for IFNb . In the present example of the anti-CD38-IFNb 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 IFNb fusion protein construct in the present example shows an
ASI of 4,630 (Table 34). The R147A mutation in IFNb , as an alternative to the R35A
mutation, was also found to produce antibody-IFNb fusion protein constructs with a
significantly greater ASI than free IFNb (data not shown). The examples below will show
that this “selective attenuation” can also be observed with ligands that are structurally
unrelated to IFNa and b , namely to IL-4 and IL-6.
9293452-1
Table 34
Test Article EC50 On EC50 Off Antigen
EC50 IFNb EC50 IFNb
(TA) Target Target (pM) Specificity
/EC50 TA /EC50 TA
(pM) (On Target; iLite (Off Target; Index
ARP-1 Column 3/
ARP-1) iLite)
Column 5
.5 1.00 0.799 1.00 1.00
IFNb
G005-HC-L0- 46.9 0.224 16,100 4.84 x 10 4,630
IFNb (R35A)
IgG4
G005-HC-L0- 32.7 0.321 57.6 0.0135 23.8
IFNb IgG4
Interleukin-4 (IL-4)
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.
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).
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Methods:
The “off-target (HB-IL4) assay” was performed largely as described by the
manufacturer of the HEK-Blue IL4/IL13 cell line. HEK-Blue™ 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™ IL-4/IL-13 Cells
stably express a reporter gene, secreted embryonic alkaline phosphatase (SEAP), under the
control of the IFNb minimal promoter fused to four STAT6 binding sites. Activation of the
STAT6 pathway in HEK-Blue™ 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™. Briefly, HEK-Blue IL4/IL13 cells (Invivogen, San
Diego CA cat# hkb-stat6) were thawed and cultured in DMEM media (Mediatech,
Manassas VA, cat# 10CV) + 10% FBS (Hyclone, Logan UT, cat# SH30070.03) that
had been heat inactivated (HI FBS). After one passage, 10 m 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# 25Cl). Cells were washed twice in
DMEM + HI FBS and counted. Cells were adjusted to 2.8 x 10 viable cells/ml in DMEM
+ HI FBS and 180 m l was aliquoted per well into a flat bottom 96 well tissue culture plate
(hereafter, the “experimental plate”). Then, 20m l of IL-4 or fusion protein construct,
diluted into DMEM + HI FBS, was added per well. The plate was incubated at 37 C 5%
CO for 16-24 hours. QUANTI-Blue (Invivogen, cat# rep-qb1) was prepared according to
the manufacturer’s directions. QUANTI-Blue (160 m l) was aliquoted into each well of a
flat bottom plate (hereafter, the “assay plate”). Then, 40 m l supernatant per well from the
experimental plate was transferred to assay plate. Assay plate was then incubated at 37 C
for 1-3 hours. Assay plate absorbance at 630nm was read on a model 1420-41 Victor 3V
Multilabel Counter from Perkin-Elmer. Data was analyzed using Graph Pad Prism.
The “on target (Th1 diversion) assay” was designed to monitor the percentage of
CD4 T cells that were of Th1 phenotype, as defined by their expression of IFN-g . Th1
diversion is thereby quantified by a decrease in IFN- g -positive CD4 T cells. The assay
was performed as follows: “Loaded” Dynabeads (M450 Epoxy beads, Invitrogen Dynal,
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Oslo, Norway cat# 140.11) were made as described by the manufacturer with 1.0 m g/10
beads anti-human CD3 epsilon antibody (R&D Systems, Minneapolis MN, cat# MAB100),
1.0 m g/10 beads anti-human CD28 antibody (R&D Systems, cat# MAB342) and 3 m g/10
beads human IgG (R&D Systems, cat# 1A). PBMCs were obtained from the
Stanford Blood Center; Palo Alto CA. Naïve CD4 T cells were purified from Leukocyte
Reduction System (LRS) cones using the naïve CD4 kit (Miltenyi Biotech cat# 130
131) according to the manufacturer’s directions. A total of 4.0 x 10 purified naïve CD4
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# 10CV) + 10% HI
FBS + 100 units/ml IL-2 (Peprotech, cat# 200-02), hereafter referred to as Media-Q. Then,
4.0 x 10 “loaded” Dynabeads were added per well. IL-12 (Peprotech, cat# 200-12) was
diluted into Media-Q and 100 m 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 m l 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 C with 5% CO 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 m g/ml. Brefeldin A was added to
a final concentration of 1.0 m g/ml of culture. The experimental plate was incubated at 37 C
with 5% CO 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# 5142). 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:
In the absence of antibody-based targeting, as measured by the “off-target (HB-
IL4) assay,” IL4 showed an EC50 of 1.26 pM (Figure 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
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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 35
Test Article EC50 On EC50 EC50 Off EC50 Antigen
(TA) Target (pM) IL4/EC50 TA Target IL4/EC50 TA Specificity
Th1 (Th1 (pM) HB- (Off Target; Index
Diversion Diversion) IL4 HB-IL4 ) Column 3/
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 x 10 8,790
IL4 (R88Q)
IgG1
Isotype-HC- >1000 ND 19,200 6.56 x 10 ND
L6-IL4
(R88Q) IgG1
The “on target (Th1 diversion) assay” results are shown in Figure 45. Activation
of the naïve (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 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
(1.45x 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.
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Interleukin-6 (IL-6)
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 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:
The “IL-6 bioassay” was performed using the HEK-Blue™ 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 IFNb 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™. 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# 10CV) + 10% FBS (Hyclone, Logan UT,
cat# SH30070.03) that had been heat inactivated (HI FBS). After one passage, 200 m g/ml
HygroGold, (Invivogen cat# ant-hg-1) and 100 m 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# 25Cl). Cells were
then washed twice in DMEM + HI FBS and counted. Cells were adjusted to 2.8 x 10
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 m l of IL-6 or fusion
protein construct, diluted into DMEM + HI FBS, was added per well. The plate was
incubated at 37 C with 5% CO for 16-24 hours. QUANTI-Blue (Invivogen, cat# rep-qb1),
prepared according to the manufacturer’s instructions, was then aliquoted (160 m l per well)
into each well of a flat bottom plate (hereafter, the “assay plate”). Then, 40 m l supernatant
per well from the experimental plate was transferred to the wells of the assay plate. The
assay plate was incubated at 37 C for 1-3 hours. Assay plate absorbance at 630nm was
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read on a model 1420-41 Victor 3V Multilabel Counter from Perkin-Elmer. Data was
analyzed using Graph Pad Prism.
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 (Figure 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™ 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 IFNα
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 IFNa 2b, were performed. In most studies this was compared to non-
targeted control fusion protein constructs referred to below as “isotype control”. The
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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).
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:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached
120-150 mm , mice were grouped into 5 cohorts of 10 mice each and treatment began at
time zero (T0). 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 m g/dose
(approximately 10 mg/kg) except for Interferon-a . IFNa 2b (Intron A®, Schering Corp.,
Merck, Whitehouse Station, NJ) was given at 2 million units/dose. Tumor volume was
measured twice weekly by caliper measurement. Endpoint was tumor volume of 2,000
mm .
Results:
Results are shown in Figure 47. Treatment of this multiple myeloma
subcutaneous (s.c.) solid tumor with interferon-a (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 ) by day 30. The non-
targeted isotype control-attenuated IFNa fusion protein construct (Isotype-HC-L6-
IFN a (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-IFNa fusion protein construct and
resulting increased systemic exposure. The CD38-targeted attenuated IFNa fusion protein
construct (G005-HC-L6-IFN a (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
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targeted anti-CD38-attenuated IFNa 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.
The anti-CD38-attenuated IFNa fusion protein construct (G005-HC-L6-
IFN a (A145G) IgG1) was tested in a systemic multiple myeloma model based on the cell
line MM1S (Crown Bioscience Inc., Santa Clara; Greenstein, Exp Hematol.
Apr;31(4):271-82,2003 ).
Methods:
Six to 8 week old NOD-SCID mice were injected intravenously with 1x10
MM1S tumor cells in 0.1 ml phosphate buffered saline (PBS) 24 hours after irradiation
with 200 rad ( Co). 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 m g/dose (approximately 10 mg/kg) except Interferon-a
(given at 2 million units/dose). Body weights and overall health were monitored twice
weekly and survival was the endpoint.
Results:
Results are shown in Figure 48. Treatment of this systemic multiple myeloma
tumor with interferon-a (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 IFNa (G005-HC-L6-IFN a (A145G) IgG1) treated cohort
showed signs of disease during entire study. All (10/10) mice appeared healthy at
termination.
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 .
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Methods:
Six to eight week old NOD-SCID mice were injected subcutaneously in the flank
with 1x10 Daudi Burkitt’s Lymphoma tumor cells in 50% Matrigel one day after
60 3
irradiation with 200rad ( Co). When mean tumor size reached 169 mm (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 m g/dose
(approximately 10 mg/kg) except Interferon-a , 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 .
Results:
Results are shown in Figure 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 IFNa treatment did result
in a significant delay in tumor growth compared to vehicle (5.5 days) however this group
reached the 2,000 mm endpoint by day 40. The non-targeted isotype control fusion
protein construct (Isotype-HC-L6-IFN a (A145G) IgG1; open inverted triangles) showed
significant activity in delaying tumor growth but this group reached the 2,000 mm
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.
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Table 36.
Median Tumor Size T/C
Treatment P value
(mm3) at Day 37 (%)
Vehicle 3034+/-340 -- --
Anti-CD38 (G005) IgG1 2443+/-196 81 0.575
0 0 <0.0001
G005-HC-L6-IFNa (145G) IgG1
Isotype-HC-L6-IFNa (145G) IgG1 15 0.5 <0.0001
IFNα 1440+/-154 47 0.007
a. Mean+/-SEM
b. Ratio of tumor size for treatment group divided by tumor size for vehicle group
at day 37
c. Vs. vehicle control at day 37
This xenograft experiment shows that the CD38-targeted attenuated IFNα fusion
protein constructs may be effective in treating lymphomas in addition to multiple
myelomas.
The effect of different doses of the anti-CD38-attenuated IFNa 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:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-
150 mm , 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 .
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Results:
Results are shown in Figure 50. The dose titration of anti-CD38-targeted
attenuated IFNa fusion protein construct (G005-HC-L6-IFNa (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-IFNa
(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.
The next example shows that antibodies of the present invention also include
those of the IgG4 isotype.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached
120-150 mm , 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 m g/dose (approximately 3.5 mg/kg). Tumor volume was measured twice
weekly by caliper measurement. Endpoint was 2,000 mm .
Results:
Results are shown in Figure 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 a (A145G) IgG1 (targeted, closed squares) and Isotype-HC-L6-
IFN a (A145G) IgG1 (non-targeted, open squares)) and IgG4 isotype (G005-HC-L6-
IFN a (A145G) IgG4, composed of SEQ ID NOS:148 (heavy chain) and 134 (light chain)
(targeted, closed diamonds) and Isotype-HC-L6-IFN a (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
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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).
The next example extends the observation of in vivo efficacy of an antibody-
targeted IFN to a second mutated form of IFNa 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 (Vl )). 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 IFNa is fused directly to the C terminus of the antibody heavy chain.
Methods:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When average tumor size reached
120-150 mm , 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 m g/dose (approximately 3 mg/kg). Tumor volume was measured twice
weekly by caliper measurement. Endpoint was tumor volume of 2,000 mm .
Results:
Results are shown in Figure 52. This anti-CD38-attenuated IFN a fusion protein
construct (X355/02-HC-L0-IFNa (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 IFNa fusion protein constructs to effectively treat human
myeloma in an in vivo model is not restricted to a single variable domain, IFNa 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.
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The next example shows that an anti-CD38 antibody-attenuated IFNa fusion
protein construct is more effective than standard drugs used to treat multiple myeloma in
the same xenograft model described above.
Methods:
[0394] Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-
150 mm , 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 .
Results:
Results are shown in Figure 53. In this study the activity of the anti-CD38
targeted fusion protein construct (G005-HC-L0-IFN a (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.
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:
Eight to 12 week old CB.17 SCID mice were injected subcutaneously in the flank
with 1x10 NCI-H929 tumor cells in 50% Matrigel. When mean tumor sizes reached 120-
150 mm mice were grouped into cohorts of 10 mice each and then treatment began (T0).
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
9293452-1
grey filled circles. Tumor volume was measured twice weekly by caliper measurement.
Endpoint was 2,000 mm .
Results:
Results are shown in Figure 54. This study surprisingly showed that a single dose
of the G005-HC-L6-IFN a (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.
The next example demonstrates that even very large tumors can be eliminated by
treatment with an anti-CD38-attenuated IFN fusion protein construct.
Methods:
One cohort (n = 9) from the immediately preceding study was not treated until the
mean tumor volume reached 730 mm . This cohort then received 6 doses of anti-CD38
targeted, attenuated interferon on days 12, 15, 19, 22, 26 and 29 (arrows).
Results:
[0401] Results are shown in Figure 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 >1000mm . The only mouse that died
from the myeloma was one which had a tumor volume of 1,800 mm at the start of
treatment; it reached the 2,000 mm 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.
It was shown that in the in vitro experiments above (Table 27) that the G005-HC-
L6-IFN a (A145G) IgG4 fusion protein construct has about 25,000-fold lower potency than
9293452-1
free, wild-type IFNa 2b under conditions where the antibody does not target the attenuated
IFNa 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 IFNa . This effect of
IFNa on hematopoiesis can be measured ex vivo by determining the effect of IFNa on the
number of colony forming units derived from primary human bone marrow mononuclear
cells. The IFNa vs. the antibody-attenuated IFNa fusion protein constructs were
compared in terms of their effect on colony formation.
Methods:
[0403] Frozen normal human bone marrow mononuclear cells (AllCells, Inc.,
Emeryville, CA) 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 1.75x10 cells/ml. Cell suspensions were
diluted with MethoCult H4434 Classic medium (Stem Cell Technologies, Cat# 04434) to a
final cell concentration of 0.7x10 cells/ml. Cells were then mixed very well and 3 ml of
this mixture was aliquoted into each tube.
Intron A (Schering Corp. Merck, NJ) and fusion protein constructs (G005-HC-L0-
IFN a (145D) IgG4 and Isotype-HC-L0-IFN a (145D) IgG4) were diluted in tenfold serial
dilutions in complete medium and 150 m 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 35mm tissue culture dish (Stem Cell Technologies, cat#27115). Plates were
then incubated in a well-humidified incubator at 37 C with 5% CO 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:
The results are shown in Figure 56. The data indicate that both the targeted (anti-
CD38, G005) and the non-targeted (isotype; 2D12) attenuated interferon fusion protein
9293452-1
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 10,000x fold less activity in inhibiting colony
formation than wild type, free IFNa , thus confirming that the A145D mutation attenuates
the IFN activity of the antibody-IFNa fusion protein constructs and suggesting that such
attenuated IFN-antibody fusion protein constructs will have a superior safety profile
compared to IFNa itself.
Another activity of IFNa that can be measured ex vivo is the stimulation of
cytokine and chemokine secretion. Normal human PBMCs were stimulated with various
concentrations of IFNa vs the antibody-attenuated IFNa fusion protein construct Isotype-
HC-L6-IFNa (A145G) IgG1 (based on the 2D12 antibody), and measured the resulting
cytokine production.
Methods:
[0407] 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 1x10 cells/ml. The cells were then incubated with
human IgG at 4 mg/ml and incubated at 37 C for 30min to block any nonspecific IgG
binding. Without washing, 250 m l aliquots of cells were then added to wells of 24-well
tissue culture treated plates. To these wells were then added 250 m l of free IFNa or an IgG-
attenuated IFNa fusion protein construct (Isotype-HC-L6-IFNa (A145G) IgG1; isotype
antibody is 2D12) at various concentrations. Plates were then incubated overnight at 37 C
in 5% CO . The following day, the plates were spun down and 200 m l of supernatant was
collected from each well. Supernatants were kept frozen until analysis using a Luminex
cytokine assay.
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
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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:
Results are shown in Figure 57. Four cytokines (IP-10, MCP-1, MCP-3 and IL-
1a ) were consistently upregulated in response to IFNa exposure. The antibody-attenuated
IFNa fusion protein construct (Isotype-HC-L6- IFNa (A145G) IgG1) showed 1,000-
,000-fold reduced potency compared to wild type IFNa 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 ~2,500-fold attenuation) and IL1a (estimated at about 1,300-fold
attenuation).
9293452-1
SEQUENCE TABLES
Table 37 - Single polypeptide chain sequences
SEQ ID
NO: Species Length Unit Gene Subtype Variant
1 human 166 aa IFN a 1b native
2 human 165 aa IFN a 2a native
3 human 165 aa IFN a 2b native
4 human 165 aa IFN a 2b L15A
human 165 aa IFN a 2b A19W
6 human 165 aa IFN a 2b R22A
7 human 165 aa IFN a 2b R23A
8 human 165 aa IFN a 2b S25A
9 human 165 aa IFN a 2b L26A
human 165 aa IFN a 2b F27A
11 human 165 aa IFN a 2b L30A
12 human 165 aa IFN a 2b L30V
13 human 165 aa IFN a 2b K31A
14 human 165 aa IFN a 2b D32A
human 165 aa IFN a 2b R33K
16 human 165 aa IFN a 2b R33A
17 human 165 aa IFN a 2b R33Q
18 human 165 aa IFN a 2b H34A
19 human 165 aa IFN a 2b Q40A
human 165 aa IFN a 2b D114R
21 human 165 aa IFN a 2b L117A
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SEQ ID
NO: Species Length Unit Gene Subtype Variant
22 human 165 aa IFN a 2b R120A
23 human 165 aa IFN a 2b R120E
24 human 165 aa IFN a 2b R125A
human 165 aa IFN a 2b R125E
26 human 165 aa IFN a 2b K131A
27 human 165 aa IFN a 2b E132A
28 human 165 aa IFN a 2b K133A
29 human 165 aa IFN a 2b K134A
human 165 aa IFN a 2b R144A
31 human 165 aa IFN a 2b R144D
32 human 165 aa IFN a 2b R144E
33 human 165 aa IFN a 2b R144G
34 human 165 aa IFN a 2b R144H
human 165 aa IFN a 2b R144I
36 human 165 aa IFN a 2b R144K
37 human 165 aa IFN a 2b R144L
38 human 165 aa IFN a 2b R144N
39 human 165 aa IFN a 2b R144Q
40 human 165 aa IFN a 2b R144S
41 human 165 aa IFN a 2b R144T
42 human 165 aa IFN a 2b R144V
43 human 165 aa IFN a 2b R144Y
44 human 165 aa IFN a 2b A145D
45 human 165 aa IFN a 2b A145E
46 human 165 aa IFN a 2b A145G
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SEQ ID
NO: Species Length Unit Gene Subtype Variant
47 human 165 aa IFN a 2b A145H
48 human 165 aa IFN a 2b A145I
49 human 165 aa IFN a 2b A145K
50 human 165 aa IFN a 2b A145L
51 human 165 aa IFN a 2b A145M
52 human 165 aa IFN a 2b A145N
53 human 165 aa IFN a 2b A145Q
54 human 165 aa IFN a 2b A145R
55 human 165 aa IFN a 2b A145S
56 human 165 aa IFN a 2b A145T
57 human 165 aa IFN a 2b A145V
58 human 165 aa IFN a 2b A145Y
59 human 165 aa IFN a 2b M148A
60 human 165 aa IFN a 2b R149A
61 human 165 aa IFN a 2b S152A
62 human 165 aa IFN a 2b L153A
63 human 165 aa IFN a 2b N156A
64 human 165 aa IFN a 2b L30A+YNS
65 human 165 aa IFN a 2b R33A+YNS
66 human 165 aa IFN a 2b M148A+YNS
67 human 165 aa IFN a 2b L153A+YNS
68 human 165 aa IFN a 2b R144A+YNS
69 human 165 aa IFN a 2b N65A,L80A,Y85A,Y89A
70 human 165 aa IFN a 2b N65A,L80A,Y85A,Y89A,D114A
71 human 165 aa IFN a 2b N65A,L80A,Y85A,Y89A,L117A
9293452-1
SEQ ID
NO: Species Length Unit Gene Subtype Variant
72 human 165 aa IFN a 2b N65A,L80A,Y85A,Y89A,R120A
73 human 165 aa IFN a 2b Y85A,Y89A,R120A
74 human 165 aa IFN a 2b D114A,R120A
75 human 165 aa IFN a 2b L117A,R120A
76 human 165 aa IFN a 2b L117A,R120A,K121A
77 human 165 aa IFN a 2b R120A,K121A
78 human 165 aa IFN a 2b R120E,K121E
79 human 160 aa IFN a 2b D [L161-E165]
80 human 166 aa IFN a 4b native
81 human 166 aa IFN a 5 native
82 human 166 aa IFN a 6 native
83 human 166 aa IFN native
84 human 166 aa IFN native
a 10
85 human 166 aa IFN native
86 human 166 aa IFN 1 a a/13 native
87 human 166 aa IFN a 14 native
88 human 166 aa IFN a 16 native
89 human 166 aa IFN a 17 native
90 human 166 aa IFN a 21 native
91 human 166 aa IFN b 1(a) native
92 human 166 aa IFN b 1(a) R27A
93 human 166 aa IFN b 1(a) R35T
94 human 166 aa IFN b 1(a) E42K
95 human 166 aa IFN b 1(a) D54N
96 human 166 aa IFN b 1(a) M62I
9293452-1
SEQ ID
NO: Species Length Unit Gene Subtype Variant
97 human 166 aa IFN b 1(a) G78S
98 human 166 aa IFN b 1(a) K123A
99 human 166 aa IFN b 1(a) C141Y
100 human 166 aa IFN b 1(a) A142T
101 human 166 aa IFN b 1(a) E149K
102 human 166 aa IFN b 1(a) R152H
103 human 166 aa IFN b 1(b) C17S
104 human 166 aa IFN b 1(b) C17S,R35A
105 human 166 aa IFN b 1(b) C17S,R147A
106 human 143 aa IFN g native
107 human 143 aa IFN S20I
108 human 143 aa IFN g S20C
109 human 143 aa IFN D21K
110 human 143 aa IFN g V22D
111 human 143 aa IFN A23Q
112 human 143 aa IFN g A23V
113 human 143 aa IFN D24A
114 human 141 aa IFN D [A23,D24]
115 human 141 aa IFN D [N25,G26]
116 human 122 aa IFN D [A123-Q143]
117 human 129 aa IFN D [K130-Q143]
118 human 132 aa IFN D [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
9293452-1
SEQ ID
NO: Species Length Unit Gene Subtype Variant
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
nucleotide
(coding ECD, for genetic
129 human 774 strand) CD38 human immunisation (DNA)
ECD, for genetic
130 human 258 aa CD38 human immunisation (translated)
131 human 300 aa CD38 human native
132 synthetic 6 aa linker 6-mer
133 synthetic 16 aa linker 16-mer
9293452-1
Table 38 – SEQ ID NOs related to proteins comprising 2 polypeptide chains
SEQ ID
NO: Protein Name Chain Species Length Unit
134 LC aa human 214 aa
135 HC aa human 452 aa
nucleotide (coding
G005 IgG1
136 LC DNA human 642 strand)
nucleotide (coding
137 HC DNA human 1356 strand)
134 LC aa human 214 aa
138 HC aa synthetic 617 aa
nucleotide (coding
G005-HC-L0-IFNa (R144A) IgG1
136 LC DNA human 642 strand)
nucleotide (coding
139 HC DNA synthetic 1851 strand)
134 LC aa human 214 aa
140 HC aa synthetic 623 aa
nucleotide (coding
G005-HC-L6-IFNa (R144A) IgG1
136 LC DNA human 642 strand)
nucleotide (coding
141 HC DNA synthetic 1869 strand)
134 LC aa human 214 aa
142 HC aa synthetic 617 aa
nucleotide (coding
G005-HC-L0-IFNa (A145G) IgG1
136 LC DNA human 642 strand)
nucleotide (coding
143 HC DNA synthetic 1851 strand)
134 LC aa human 214 aa
144 HC aa synthetic 623 aa
nucleotide (coding
G005-HC-L6-IFNa (A145G) IgG1
136 LC DNA human 642 strand)
nucleotide (coding
145 HC DNA synthetic 1869 strand)
134 LC aa human 214 aa
146 HC aa synthetic 620 aa
nucleotide (coding
G005-HC-L6-IFNa (R144A) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
147 HC DNA synthetic 1860 strand)
134 LC aa human 214 aa
148 HC aa synthetic 620 aa
nucleotide (coding
G005-HC-L6-IFNa (A145G) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
149 HC DNA synthetic 1860 strand)
134 LC aa human 214 aa
150 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa IgG4
136 LC DNA human 642 strand)
nucleotide (coding
151 HC DNA synthetic 1842 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
134 LC aa human 214 aa
152 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144A) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
153 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
154 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144D) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
155 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
156 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144E) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
157 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
158 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144G) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
159 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
160 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144H) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
161 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
162 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144I) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
163 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
164 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144K) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
165 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
166 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144L) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
167 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
G005-HC-L0-IFNa (R144N) IgG4
168 HC aa synthetic 614 aa
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
136 LC DNA human 642 strand)
nucleotide (coding
169 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
170 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144Q) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
171 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
172 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144S) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
173 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
174 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144T) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
175 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
176 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144V) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
177 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
178 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (R144Y) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
179 HC DNA synthetic 1842 strand)
G005-HC-L0-IFNa (A145D) IgG4
134 LC aa human 214 aa
180 HC aa synthetic 614 aa
nucleotide (coding
136 LC DNA human 642 strand)
nucleotide (coding
181 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
182 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145E) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
183 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
184 HC aa synthetic 614 aa
G005-HC-L0-IFNa (A145G) IgG4
nucleotide (coding
136 LC DNA human 642 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
185 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
186 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145H) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
187 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
188 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145I) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
189 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
190 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145K) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
191 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
192 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145L) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
193 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
194 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145N) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
195 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
196 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145Q) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
197 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
198 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145R) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
199 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
200 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145S) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
201 HC DNA synthetic 1842 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
134 LC aa human 214 aa
202 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145T) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
203 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
204 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145V) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
205 HC DNA synthetic 1842 strand)
134 LC aa human 214 aa
206 HC aa synthetic 614 aa
nucleotide (coding
G005-HC-L0-IFNa (A145Y) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
207 HC DNA synthetic 1842 strand)
208 LC aa synthetic 385 aa
135 HC aa human 452 aa
nucleotide (coding
G005-LC-L6-IFNa (A145G) IgG1
209 LC DNA synthetic 1155 strand)
nucleotide (coding
137 HC DNA human 1356 strand)
210 LC aa synthetic 379 aa
135 HC aa human 452 aa
nucleotide (coding
G005-LC-L0-IFNa (A145G) IgG1
211 LC DNA synthetic 1137 strand)
nucleotide (coding
137 HC DNA human 1356 strand)
134 LC aa human 214 aa
212 HC aa synthetic 615 aa
nucleotide (coding
G005-HC-L0-IFNb IgG4
136 LC DNA human 642 strand)
nucleotide (coding
213 HC DNA synthetic 1845 strand)
134 LC aa human 214 aa
214 HC aa synthetic 615 aa
nucleotide (coding
G005-HC-L0-IFNb (R35A) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
215 HC DNA synthetic 1845 strand)
134 LC aa human 214 aa
216 HC aa synthetic 615 aa
nucleotide (coding
G005-HC-L0-IFNb (R147A) IgG4
136 LC DNA human 642 strand)
nucleotide (coding
217 HC DNA synthetic 1845 strand)
218 LC aa human 212 aa
MORAB03080 IgG1
219 HC aa human 452 aa
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
220 LC DNA human 636 strand)
nucleotide (coding
221 HC DNA human 1356 strand)
222 LC aa synthetic 214 aa
223 HC aa synthetic 450 aa
nucleotide (coding
hu38SB19 (SAR650984) IgG1
224 LC DNA synthetic 642 strand)
nucleotide (coding
225 HC DNA synthetic 1350 strand)
226 LC aa human 222 aa
227 HC aa human 451 aa
nucleotide (coding
X355/02 IgG1
228 LC DNA human 666 strand)
nucleotide (coding
229 HC DNA human 1353 strand)
226 LC aa human 222 aa
230 HC aa synthetic 613 aa
nucleotide (coding
X355/02-HC-L0-IFNa (R144A) IgG4
228 LC DNA human 666 strand)
nucleotide (coding
231 HC DNA synthetic 1839 strand)
226 LC aa human 222 aa
232 HC aa synthetic 613 aa
nucleotide (coding
X355/02-HC-L0-IFNa (A145D) IgG4
228 LC DNA human 666 strand)
nucleotide (coding
233 HC DNA synthetic 1839 strand)
234 LC aa human 215 aa
235 HC aa human 448 aa
nucleotide (coding
X355/07 IgG
236 LC DNA human 645 strand)
nucleotide (coding
237 HC DNA human 1344 strand)
234 LC aa human 215 aa
238 HC aa synthetic 610 aa
nucleotide (coding
X355/07-HC-L0-IFNa (R144A) IgG4
236 LC DNA human 645 strand)
nucleotide (coding
239 HC DNA synthetic 1830 strand)
234 LC aa human 215 aa
240 HC aa synthetic 610 aa
nucleotide (coding
X355/07-HC-L0-IFNa (A145D) IgG4
236 LC DNA human 645 strand)
nucleotide (coding
241 HC DNA synthetic 1830 strand)
242 LC aa human 222 aa
243 HC aa human 452 aa
X910/12 IgG1
nucleotide (coding
244 LC DNA human 666 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
245 HC DNA human 1356 strand)
242 LC aa human 222 aa
246 HC aa synthetic 614 aa
nucleotide (coding
X910/12-HC-L0-IFNa (R144A) IgG4
244 LC DNA human 666 strand)
nucleotide (coding
247 HC DNA synthetic 1842 strand)
242 LC aa human 222 aa
248 HC aa synthetic 614 aa
nucleotide (coding
X910/12-HC-L0-IFNa (A145D) IgG4
244 LC DNA human 666 strand)
nucleotide (coding
249 HC DNA synthetic 1842 strand)
250 LC aa human 222 aa
251 HC aa human 450 aa
nucleotide (coding
X913/15 IgG1
252 LC DNA human 666 strand)
nucleotide (coding
253 HC DNA human 1350 strand)
250 LC aa human 222 aa
254 HC aa synthetic 612 aa
nucleotide (coding
X913/15-HC-L0-IFNa (R144A) IgG4
252 LC DNA human 666 strand)
nucleotide (coding
255 HC DNA synthetic 1836 strand)
250 LC aa human 222 aa
256 HC aa synthetic 612 aa
nucleotide (coding
X913/15-HC-L0-IFNa (A145D) IgG4
252 LC DNA human 666 strand)
nucleotide (coding
257 HC DNA synthetic 1836 strand)
258 LC aa synthetic 214 aa
259 HC aa synthetic 450 aa
nucleotide (coding
R5D1 IgG1
260 LC DNA synthetic 642 strand)
nucleotide (coding
261 HC DNA synthetic 1350 strand)
258 LC aa synthetic 214 aa
262 HC aa synthetic 612 aa
nucleotide (coding
R5D1-HC-L0-IFNa (A145D) IgG4
260 LC DNA synthetic 642 strand)
nucleotide (coding
263 HC DNA synthetic 1836 strand)
264 LC aa synthetic 219 aa
265 HC aa synthetic 453 aa
nucleotide (coding
R5E8 IgG1
266 LC DNA synthetic 657 strand)
nucleotide (coding
267 HC DNA synthetic 1359 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
264 LC aa synthetic 219 aa
268 HC aa synthetic 615 aa
nucleotide (coding
R5E8-HC-L0-IFNa (A145D) IgG4
266 LC DNA synthetic 657 strand)
nucleotide (coding
269 HC DNA synthetic 1845 strand)
270 LC aa synthetic 214 aa
271 HC aa synthetic 450 aa
nucleotide (coding
R10A2 IgG1
272 LC DNA synthetic 642 strand)
nucleotide (coding
273 HC DNA synthetic 1350 strand)
270 LC aa synthetic 214 aa
274 HC aa synthetic 612 aa
nucleotide (coding
R10A2-HC-L0-IFNa (A145D) IgG4
272 LC DNA synthetic 642 strand)
nucleotide (coding
275 HC DNA synthetic 1836 strand)
276 LC aa synthetic 213 aa
277 HC aa synthetic 451 aa
nucleotide (coding
Rituximab
278 LC DNA synthetic 639 strand)
nucleotide (coding
279 HC DNA synthetic 1353 strand)
276 LC aa synthetic 213 aa
280 HC aa synthetic 622 aa
nucleotide (coding
Rituximab-HC-L6-IFNa IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
281 HC DNA synthetic 1866 strand)
276 LC aa synthetic 213 aa
282 HC aa synthetic 622 aa
Rituximab-HC-L6-IFNa (R144A)
nucleotide (coding
IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
283 HC DNA synthetic 1866 strand)
276 LC aa synthetic 213 aa
284 HC aa synthetic 622 aa
Rituximab-HC-L6-IFNa (A145G)
nucleotide (coding
IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
285 HC DNA synthetic 1866 strand)
276 LC aa synthetic 213 aa
286 HC aa synthetic 622 aa
Rituximab-HC-L6-IFNa (R33A+YNS)
nucleotide (coding
IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
287 HC DNA synthetic 1866 strand)
276 Rituximab-HC-L6- LC aa synthetic 213 aa
IFNa (R144A+YNS) IgG1
288 HC aa synthetic 622 aa
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
278 LC DNA synthetic 639 strand)
nucleotide (coding
289 HC DNA synthetic 1866 strand)
290 LC aa synthetic 213 aa
291 HC aa synthetic 450 aa
nucleotide (coding
Palivizumab
292 LC DNA synthetic 639 strand)
nucleotide (coding
293 HC DNA synthetic 1350 strand)
290 LC aa synthetic 213 aa
294 HC aa synthetic 621 aa
nucleotide (coding
Palivizumab-HC-L6-IFNa IgG1
292 LC DNA synthetic 639 strand)
nucleotide (coding
295 HC DNA synthetic 1863 strand)
290 LC aa synthetic 213 aa
296 HC aa synthetic 394 aa
nucleotide (coding
Palivizumab-HC-L6-IFNa Fab
292 LC DNA synthetic 639 strand)
nucleotide (coding
297 HC DNA synthetic 1182 strand)
290 LC aa synthetic 213 aa
298 HC aa synthetic 394 aa
Palivizumab-HC-L6-IFNa (A145D)
nucleotide (coding
292 LC DNA synthetic 639 strand)
nucleotide (coding
299 HC DNA synthetic 1182 strand)
300 LC aa synthetic 214 aa
301 HC aa synthetic 449 aa
nucleotide (coding
J110 IgG1
302 LC DNA synthetic 642 strand)
nucleotide (coding
303 HC DNA synthetic 1347 strand)
300 LC aa synthetic 214 aa
304 HC aa synthetic 584 aa
nucleotide (coding
J110-HC-L6-IL-4 IgG1
302 LC DNA synthetic 642 strand)
nucleotide (coding
305 HC DNA synthetic 1752 strand)
300 LC aa synthetic 214 aa
306 HC aa synthetic 584 aa
nucleotide (coding
J110-HC-L6-IL-4(R88Q) IgG1
302 LC DNA synthetic 642 strand)
nucleotide (coding
307 HC DNA synthetic 1752 strand)
300 LC aa synthetic 214 aa
308 HC aa synthetic 649 aa
J110-HC-L16-IL-6 IgG1
nucleotide (coding
302 LC DNA synthetic 642 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
309 HC DNA synthetic 1947 strand)
300 LC aa synthetic 214 aa
310 HC aa synthetic 649 aa
nucleotide (coding
J110-HC-L16-IL-6(R179E) IgG1
302 LC DNA synthetic 642 strand)
nucleotide (coding
311 HC DNA synthetic 1947 strand)
312 LC aa synthetic 215 aa
313 HC aa synthetic 450 aa
nucleotide (coding
HB95 IgG1
314 LC DNA synthetic 645 strand)
nucleotide (coding
315 HC DNA synthetic 1350 strand)
312 LC aa synthetic 215 aa
316 HC aa synthetic 612 aa
nucleotide (coding
HB95-HC-L0-IFNa (A145D) IgG4
314 LC DNA synthetic 645 strand)
nucleotide (coding
317 HC DNA synthetic 1836 strand)
312 LC aa synthetic 215 aa
318 HC aa synthetic 394 aa
nucleotide (coding
HB95-HC-L6-IFNa Fab
314 LC DNA synthetic 645 strand)
nucleotide (coding
319 HC DNA synthetic 1182 strand)
312 LC aa synthetic 215 aa
320 HC aa synthetic 394 aa
nucleotide (coding
HB95-HC-L6-IFNa (A145D) Fab
314 LC DNA synthetic 645 strand)
nucleotide (coding
321 HC DNA synthetic 1182 strand)
312 LC aa synthetic 215 aa
322 HC aa synthetic 650 aa
nucleotide (coding
HB95-HC-L16-IL-6 IgG1
314 LC DNA synthetic 645 strand)
nucleotide (coding
323 HC DNA synthetic 1950 strand)
312 LC aa synthetic 215 aa
324 HC aa synthetic 650 aa
nucleotide (coding
HB95-HC-L16-IL-6(R179E) IgG1
314 LC DNA synthetic 645 strand)
nucleotide (coding
325 HC DNA synthetic 1950 strand)
326 LC aa synthetic 214 aa
327 HC aa synthetic 452 aa
nucleotide (coding
nBT062 IgG1
328 LC DNA synthetic 642 strand)
nucleotide (coding
329 HC DNA synthetic 1356 strand)
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
326 LC aa synthetic 214 aa
330 HC aa synthetic 614 aa
nucleotide (coding
nBT062-HC-L0-IFNa (A145D) IgG4
328 LC DNA synthetic 642 strand)
nucleotide (coding
331 HC DNA synthetic 1842 strand)
332 LC aa synthetic 214 aa
333 HC aa synthetic 448 aa
nucleotide (coding
C21 IgG1
334 LC DNA synthetic 642 strand)
nucleotide (coding
335 HC DNA synthetic 1344 strand)
332 LC aa synthetic 214 aa
336 HC aa synthetic 610 aa
nucleotide (coding
C21-HC-L0-IFNa (A145D) IgG4
334 LC DNA synthetic 642 strand)
nucleotide (coding
337 HC DNA synthetic 1830 strand)
338 LC aa synthetic 214 aa
339 HC aa synthetic 449 aa
nucleotide (coding
7.1 IgG1
340 LC DNA synthetic 642 strand)
nucleotide (coding
341 HC DNA synthetic 1347 strand)
338 LC aa synthetic 214 aa
342 HC aa synthetic 611 aa
nucleotide (coding
7.1-HC-L0-IFNa (A145D) IgG4
340 LC DNA synthetic 642 strand)
nucleotide (coding
343 HC DNA synthetic 1833 strand)
344 LC aa synthetic 213 aa
345 HC aa synthetic 452 aa
nucleotide (coding
2D12 IgG1
346 LC DNA synthetic 639 strand)
nucleotide (coding
347 HC DNA synthetic 1356 strand)
344 LC aa synthetic 213 aa
348 HC aa synthetic 623 aa
nucleotide (coding
2D12-HC-L6-IFNa (A145G) IgG1
346 LC DNA synthetic 639 strand)
nucleotide (coding
349 HC DNA synthetic 1869 strand)
344 LC aa synthetic 213 aa
350 HC aa synthetic 620 aa
nucleotide (coding
2D12-HC-L6-IFNa (A145G) IgG4
346 LC DNA synthetic 639 strand)
nucleotide (coding
351 HC DNA synthetic 1860 strand)
344 LC aa synthetic 213 aa
2D12-HC-L0-IFNa (A145D) IgG4
352 HC aa synthetic 614 aa
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
nucleotide (coding
346 LC DNA synthetic 639 strand)
nucleotide (coding
353 HC DNA synthetic 1842 strand)
344 LC aa synthetic 213 aa
354 HC aa synthetic 396 aa
nucleotide (coding
2D12-HC-L6-IFNa Fab
346 LC DNA synthetic 639 strand)
nucleotide (coding
355 HC DNA synthetic 1188 strand)
344 LC aa synthetic 213 aa
356 HC aa synthetic 396 aa
nucleotide (coding
2D12-HC-L6-IFNa (A145D) Fab
346 LC DNA synthetic 639 strand)
nucleotide (coding
357 HC DNA synthetic 1188 strand)
344 LC aa synthetic 213 aa
358 HC aa synthetic 587 aa
nucleotide (coding
2D12-HC-L6-IL-4(R88Q) IgG1
346 LC DNA synthetic 639 strand)
nucleotide (coding
359 HC DNA synthetic 1761 strand)
344 LC aa synthetic 213 aa
360 HC aa synthetic 652 aa
nucleotide (coding
2D12-HC-L16-IL-6 IgG1
346 LC DNA synthetic 639 strand)
nucleotide (coding
361 HC DNA synthetic 1956 strand)
344 LC aa synthetic 213 aa
362 HC aa synthetic 652 aa
nucleotide (coding
2D12-HC-L16-IL-6(R179E) IgG1
346 LC DNA synthetic 639 strand)
nucleotide (coding
363 HC DNA synthetic 1956 strand)
364 LC aa human 214 aa
365 HC aa human 455 aa
nucleotide (coding
X355/01 IgG1
366 LC DNA human 642 strand)
nucleotide (coding
367 HC DNA human 1365 strand)
368 LC aa human 219 aa
369 HC aa human 453 aa
nucleotide (coding
X355/04 IgG1
370 LC DNA human 657 strand)
nucleotide (coding
371 HC DNA human 1359 strand)
372 LC aa synthetic 220 aa
R10B10 IgG1
373 HC aa synthetic 449 aa
374 LC aa synthetic 220 aa
R7H11 IgG1
375 HC aa synthetic 449 aa
9293452-1
SEQ ID
NO: Protein Name Chain Species Length Unit
376 LC aa synthetic 214 aa
R7F11 IgG1
377 HC aa synthetic 452 aa
276 LC aa synthetic 213 aa
378 HC aa synthetic 599 aa
Rituximab-HC-L7-IFNg (D[ A23,D24])
nucleotide (coding
IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
379 HC DNA synthetic 1797 strand)
226 LC aa human 222 aa
380 HC aa synthetic 601 aa
nucleotide (coding
X355/02-HC-L7-IFNg (S20I) IgG1
228 LC DNA human 666 strand)
nucleotide (coding
381 HC DNA synthetic 1803 strand)
270 LC aa synthetic 214 aa
382 HC aa synthetic 600 aa
nucleotide (coding
R10A2-HC-L7-IFNg (D21K) IgG1
272 LC DNA synthetic 642 strand)
nucleotide (coding
383 HC DNA synthetic 1800 strand)
276 LC aa synthetic 213 aa
436 HC aa synthetic 622 aa
nucleotide (coding
Rituximab-HC-L6-IFNa (R33A) IgG1
278 LC DNA synthetic 639 strand)
nucleotide (coding
437 HC DNA synthetic 1866 strand)
9293452-1
Table 39 – Variable Domains
SEQ ID
NO: Clone Antigen Species Length (aa)
Chain
384 G005 CD38 human 107
385 G005 CD38 human 122
386 MORAB03080 CD38 human 106
387 MORAB03080 CD38 human 122
hu38SB19
CD38
388 (SAR650984) synthetic 107
hu38SB19
CD38
389 (SAR650984) synthetic 120
390 X355/02 CD38 human 116
391 X355/02 CD38 human 121
392 X355/07 CD38 human 108
393 X355/07 CD38 human 118
394 X910/12 CD38 human 116
395 X910/12 CD38 human 122
396 X913/15 CD38 human 116
397 X913/15 CD38 human 120
398 R5D1 CD38 rat 107
399 R5D1 CD38 rat 120
400 R5E8 CD38 rat 112
401 R5E8 CD38 rat 123
402 R10A2 CD38 rat 107
403 R10A2 CD38 rat 120
404 Rituximab CD20 mouse 106
405 Rituximab CD20 mouse 121
9293452-1
SEQ ID
NO: Clone Antigen Species Length (aa)
Chain
Respiratory
Palivizumab Syncytial Virus
406 (RSV) synthetic 106
407 Palivizumab RSV synthetic 120
408 J110 PD-1 mouse 107
409 J110 PD-1 mouse 119
410 HB95 HLA mouse 108
411 HB95 HLA mouse 120
412 nBT062 CD138 mouse 107
413 nBT062 CD138 mouse 122
High Molecular
Weight
Melanoma-
Associated
Antigen
414 (HMW-MAA) synthetic 108
415 C21 HMW-MAA synthetic 118
416 7.1 HMW-MAA mouse 107
417 7.1 HMW-MAA mouse 119
Yellow Fever
2D12
418 Virus (YFV) mouse 106
419 2D12 YFV mouse 122
420 X355/01 CD38 human 107
421 X355/01 CD38 human 125
422 X355/04 CD38 human 112
423 X355/04 CD38 human 123
424 R10B10 CD38 rat 114
425 R10B10 CD38 rat 119
9293452-1
SEQ ID
NO: Clone Antigen Species Length (aa)
Chain
426 R7H11 CD38 rat 114
427 R7H11 CD38 rat 119
428 R7F11 CD38 rat 107
429 R7F11 CD38 rat 122
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
High Molecular Weight Melanoma-Associated
433 human 2322 Antigen (HMW-MAA)
434 human 165 IFNa 2c
435 human 166 IFNa 4a
9293452-1
Claims (28)
1. 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 and wherein the peptide or polypeptide signaling ligand is selected from the group consisting of an IFN, IL-4 and IL-6.
2. The polypeptide construct as claimed in claim 1 in which the peptide or polypeptide signaling ligand is linked to the antibody or antigen binding portion thereof via a peptide bond.
3. The polypeptide construct as claimed in claim 1 or claim 2 in which the peptide or polypeptide signaling ligand is linked to the antibody or antigen binding portion thereof directly or via a linker of 1 to 20 amino acids in length.
4. The polypeptide construct as claimed in any one of claims 1 to 3 in which the peptide or polypeptide signaling ligand is linked to the C-terminus of the light chain or heavy chain constant region of the antibody or antigen binding portion thereof.
5. The polypeptide construct as claimed in any one of claims claim 1 to 4 in which peptide or polypeptide signaling ligand is selected from the group consisting of an IFNa , an IFNb , and an IFNg .
6. The polypeptide construct as claimed in claim 5, in which the amino acid sequence of the IFNa is selected from SEQ ID NOs 1 to 3, 80 to 90, 434 and 435 and wherein the IFNa comprises at least one amino acid substitution or deletion which reduces its potency on cells lacking expression of said antigen.
7. The polypeptide construct as claimed in claim 6, in which the amino acid sequence of the IFNa comprising at least one amino acid 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), 9293452-1 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).
8. The polypeptide construct as claimed in any one of claims 1 to 7 in which the antibody or antigen binding portion thereof binds an antigen wherein the extracellular domain thereof has a molecular weight of less than 240kD.
9. The polypeptide construct as claimed in any one of claims 1 to 8 in which the antibody or antigen binding portion thereof binds an antigen wherein the antigen is present on the cell at a density of greater than 12,600 copies per cell or greater than 15,000 copies per cell.
10. The polypeptide construct as claimed in any one of claims 1 to 9 in which the antibody or antigen binding portion thereof binds to the cell surface-associated antigen with an affinity of from 50 nM, from 25 nM, from 10 nM, or from 5 nM to 1pM.
11. The polypeptide construct as claimed in any one of claims claim 1 to 10 in which the the cell surface-associated antigen is selected from the group consisting of CD38, HM1.24, CD56, CS1, CD20, CD74, IL-6R, Blys (BAFF), BCMA, HLA- SR, Kininogen, beta2 microglobulin, FGFR3, ICAM-1, matriptase, CD52, EGFR, GM2, alpha4-integrin, IFG-1R, KIR, CD3, CD4, CD8, CD24, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, CD11c, CD14, CD52, CD14, FSP1, FAP, PDGFR alpha, PDGFR beta, ASGR1, ASGR2, FSP1, RTI140/Ti-alpha, HTI56, VEGF receptor, 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.
12. The polypeptide construct as claimed in any one of claims 1 to 11 in which the antibody or antigen binding portion thereof is an antibody or an Fab fragment.
13. The polypeptide construct as claimed in any one of claims 1 to 12 in which the construct has an Antigen-Specificity Index of greater than 50. 9293452-1
14. The polypeptide construct as claimed in claim 13 in which the construct has an Antigen-Specificity Index of greater than 100.
15. The polypeptide construct as claimed in claim 14 in which the construct has an Antigen-Specificity Index of greater than 1000.
16. The polypeptide construct as claimed in claim 5 wherein the peptide or polypeptide signaling ligand is IFNβ and the amino acid substitution is R35A.
17. A composition comprising the polypeptide construct as claimed in any one of claims 1 to 16 and a pharmaceutically acceptable carrier or diluent.
18. The use of the polypeptide construct as claimed in any one of claims 1 to 16 in the preparation of a medicament for treatment of cancer.
19. The use as claimed in claim 18 wherein the cancer is multiple myeloma or non- Hodgkin’s lymphoma.
20. A 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; wherein the peptide or polypeptide signaling ligand is selected from the group consisting of an IFN, IL-4 and IL-6.
21. The method as claimed in claim 20 in which the peptide or polypeptide signaling ligand is linked to the antibody or antigen binding portion thereof via a peptide bond. 9293452-1
22. The method as claimed in claim 20 or claim 21 in which the peptide or polypeptide signaling ligand is linked to the antibody or antigen binding portion thereof directly or via a linker of 1 to 20 amino acids in length.
23. The method as claimed in any one of claims claim 20 to 22 in which peptide or polypeptide signaling ligand is selected from the group consisting of an IFNa , an IFNb , and an IFNg .
24. The method as claimed in claim 23, in which the amino acid sequence of the IFNa prior to modification is selected from SEQ ID NOs 1 to 3, 80 to 90, 434 and 435.
25. The method as claimed in claim 24, in which the modification comprises at least one amino acid substitution 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).
26. The method as claimed in any one of claims 20 to 25 in which the antibody or antigen binding portion thereof binds to the cell surface-associated antigen with an affinity of from 50 nM, from 25 nM, from 10 nM, or from 5 nM to 1pM.
27. The method as claimed in any one of claims claim 20 to 26 in which the the cell surface-associated antigen is selected from the group consisting of CD38, HM1.24, CD56, CS1, CD20, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR, Kininogen, beta2 microglobulin, FGFR3, ICAM-1, matriptase, CD52, EGFR, GM2, alpha4-integrin, IFG-1R, KIR, CD3, CD4, CD8, CD24, CD44, CD69, CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, CD11c, CD14, CD52, CD14, FSP1, FAP, PDGFR alpha, PDGFR beta, ASGR1, ASGR2, FSP1, RTI140/Ti-alpha, HTI56, VEGF receptor, 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. 9293452-1
28. The method as claimed in any one of claims 20 to 27 in which the antibody or antigen binding portion thereof is an antibody or an Fab fragment. 9293452-1
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ715807A NZ715807B2 (en) | 2011-10-28 | 2012-10-29 | Polypeptide constructs and uses thereof |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2011904502A AU2011904502A0 (en) | 2011-10-28 | Polypeptide constructs and uses thereof | |
AU2011904502 | 2011-10-28 | ||
PCT/AU2012/001323 WO2013059885A2 (en) | 2011-10-28 | 2012-10-29 | Polypeptide constructs and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ623812A NZ623812A (en) | 2016-02-26 |
NZ623812B2 true NZ623812B2 (en) | 2016-05-27 |
Family
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